Psma ligands for imaging and endoradiotherapy

ABSTRACT

The present invention relates to compounds which bind and/or inhibit prostate-specific membrane antigen (PSMA) comprising at least one group electron dense substituent (EDS), and at least one moiety which is amenable to radiolabeling; and therapeutic and diagnostic uses thereof.

The present disclosure relates to imaging and endoradiotherapy ofdiseases involving prostate-specific membrane antigen (PSMA). Providedare compounds which bind or inhibit PSMA and furthermore carry at leastone moiety which is amenable to radiolabeling. Provided are also medicaluses of such compounds.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Prostate Cancer (PCa) remained over the last decades the most commonmalignant disease in men with high incidence for poor survival rates.Due to its overexpression in prostate cancer (Silver, D. A., et al.,Prostate-specific membrane antigen expression in normal and malignanthuman tissues. Clinical Cancer Research, 1997. 3(1): p. 81-85),prostate-specific membrane antigen (PSMA) or glutamate carboxypeptidaseII (GCP II) proved its eligibility as excellent target for thedevelopment of highly sensitive radiolabeled agents for endoradiotherapyand imaging of PCa (Afshar-Oromieh, A., et al., The diagnostic value ofPET/CT imaging with the 68Ga-labelled PSMA ligand HBED-CC in thediagnosis of recurrent prostate cancer. European journal of nuclearmedicine and molecular imaging, 2015. 42(2): p. 197-209; Benešová, M.,et al., Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMAInhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapyof Prostate Cancer. Journal of Nuclear Medicine, 2015. 56(6): p.914-920; Robu, S., et al., Preclinical evaluation and first patientapplication of 99mTc-PSMA-I&S for SPECT imaging and radioguided surgeryin prostate cancer. Journal of Nuclear Medicine, 2016: p. jnumed.116.178939; Weineisen, M., et al., Development and first in humanevaluation of PSMA I&T-A ligand for diagnostic imaging andendoradiotherapy of prostate cancer. Journal of Nuclear Medicine, 2014.55(supplement 1): p. 1083-1083; Rowe, S., et al., PET imaging ofprostate-specific membrane antigen in prostate cancer: current state ofthe art and future challenges. Prostate cancer and prostatic diseases,2016; Maurer, T., et al., Current use of PSMA-PET in prostate cancermanagement. Nature Reviews Urology, 2016). Prostate-specific membraneantigen is an extracellular hydrolase whose catalytic center comprisestwo zinc(II) ions with a bridging hydroxido ligand. It is highlyupregulated in metastatic and hormone-refractory prostate carcinomas,but its physiologic expression has also been reported in kidneys,salivary glands, small intestine, brain and, to a low extent, also inhealthy prostate tissue. In the intestine, PSMA facilitates absorptionof folate by conversion of pteroylpoly-γ-glutamate to thepteroylglutamate (folate). In the brain, it hydrolysesN-acetyl-Laspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate andglutamate. The enzymatic function of PSMA in normal and diseasedprostate has yet not been clarified.

PSMA targeting molecules usually comprise a binding unit thatencompasses a zinc-binding group (such as urea (Zhou, J., et al., NAAGpeptidase inhibitors and their potential for diagnosis and therapy.Nature Reviews Drug Discovery, 2005. 4(12): p. 1015-1026), phosphinateor phosphoramidate) connected to a P1′ glutamate moiety, which warrantshigh affinity and specificity to PSMA and is typically further connectedto an effector functionality (Machulkin, A. E., et al., Small-moleculePSMA ligands. Current state, SAR and perspectives. Journal of drugtargeting, 2016: p. 1-15). The effector part is more flexible and tosome extent tolerant towards structural modifications. The entrancetunnel of PSMA accommodates two other prominent structural features,which are important for ligand binding. The first one is an argininepatch, a positively charged area at the wall of the entrance funnel andthe structural explanation for the preference of negatively chargedfunctionalities at the P1 position of PSMA. Upon binding the concertedrepositioning of the arginine side chains can lead to the opening of anS1 hydrophobic accessory pocket, the second important structure, thathas been shown to accommodate an iodo-benzyl group of several urea basedinhibitors, thus contributing to their high affinity for PSMA (Barinka,C., et al., Interactions between Human Glutamate Carboxypeptidase II andUrea-Based Inhibitors: Structural Characterization †. Journal ofmedicinal chemistry, 2008. 51(24): p. 7737-7743).

Zhang et al. discovered a remote binding site of PSMA, which can beemployed for bidentate binding mode (Zhang, A. X., et al., A remotearene-binding site on prostate specific membrane antigen revealed byantibody-recruiting small molecules. Journal of the American ChemicalSociety, 2010. 132(36): p. 12711-12716). The so called arene-bindingsite is a simple structural motif shaped by the side chains of Arg463,Arg511 and Trp541, and is part of the PSMA entrance lid. The engagementof the arene-binding site by a distal inhibitor moiety can result in asubstantial increase in the inhibitor affinity for PSMA due to avidityeffects. PSMA I&T (see FIG. 1 ) was developed with the intention tointeract this way with PSMA, albeit no crystal structure analysis ofbinding mode is available. A necessary feature according to Zhang et al.is a linker unit (suberic acid in the case of PSMA I&T) whichfacilitates an open conformation of the entrance lid of PSMA and therebyenabling the accessibility of the arene-binding site. It was furthershown that the structural composition of the linker has a significantimpact on the tumor-targeting and biologic activity as well as onimaging contrast and pharmacokinetics (Liu, T., et al., Spacer lengtheffects on in vitro imaging and surface accessibility of fluorescentinhibitors of prostate specific membrane antigen. Bioorganic & medicinalchemistry letters, 2011. 21(23): p. 7013-7016), properties which arecrucial for both high imaging quality and efficient targetedendoradiotherapy.

Two categories of PSMA targeting inhibitors are currently used inclinical settings. On the one side are tracer with chelating units forradionuclide complexation as PSMA I&T or related compounds (Kiess, A.P., et al., Prostate-specific membrane antigen as a target for cancerimaging and therapy. The quarterly journal of nuclear medicine andmolecular imaging: official publication of the Italian Association ofNuclear Medicine (AIMN) [and] the International Association ofRadiopharmacology (IAR), [and] Section of the Society of . . . 2015.59(3): p. 241). On the other side are small molecules, comprising atargeting unit and effector molecules. Depending on the usedradionuclide/halogen, the radiolabeled PSMA inhibitors may be used forimaging or endoradiotherapy. Among small molecule inhibitors withchelators for imaging, the most often used agents for selective PSMAimaging are PSMA HBED-CC (Eder, M., et al., 68Ga-complex lipophilicityand the targeting property of a urea-based PSMA inhibitor for PETimaging. Bioconjugate chemistry, 2012. 23(4): p. 688-697), PSMA-617(Benešová, M., et al., Preclinical Evaluation of a Tailor-MadeDOTA-Conjugated PSMA inhibitor with Optimized Linker Moiety for Imagingand Endoradiotherapy of Prostate Cancer. Journal of Nuclear Medicine,2015. 56(6): p. 914-920) and PSMA I&T (Weineisen, M., et al.,Development and first in human evaluation of PSMA I&T—A ligand fordiagnostic imaging and endoradiotherapy of prostate cancer. Journal ofNuclear Medicine, 2014. 55(supplement 1): p. 1083-1083). PSMA HBED-CC,or PSMA-11 was one the first PSMA inhibitors and is currently used forimaging since therapeutic applications are not possible with thechelator HBED-CC. However, due to the unique physical characteristicsand the advantages of 18F for PET imaging, like the longer half-life,the low positron energy, which results in higher image resolution andthe possibility for largescale production in a cyclotron, several groupshave focused on the development of ¹⁸F-labeled urea-based Inhibitors forPCa imaging. The ¹⁸F-labeled urea-based PSMA inhibitor [¹⁸F]DCFPyldemonstrated promising results in detection of primary and metastaticPCa (Rowe, S. P., et al., PSMA-Based [18F] DCFPyL PET/CT Is Superior toConventional Imaging for Lesion Detection in Patients with MetastaticProstate Cancer. Molecular Imaging and Biology, 2016: p. 1-9) andsuperiority to [⁶⁸Ga]PSMA-HBED-CC in a comparative study (Dietlein, M.,et al., Comparison of [18F] DCFPyL and [68Ga] Ga-PSMA-HBED-CC forPSMA-PET imaging in patients with relapsed prostate cancer. MolecularImaging and Biology, 2015. 17(4): p. 575-584).

PSMA DKFZ 617 (Benešová, M., et al., Preclinical Evaluation of aTailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moietyfor Imaging and Endoradiotherapy of Prostate Cancer. Journal of NuclearMedicine, 2015. 56(6): p. 914-920, Becker, A., et al., Nephro-andhepatotoxicity after radioligand therapy of metastaticcastrate-resistant prostate cancer with 177Lu-PSMA-617. Journal ofNuclear Medicine, 2016. 57(supplement 2): p. 1430-1430; Rahbar, K., etal., Response and tolerability of a single dose of 177Lu-PSMA-617 inpatients with metastatic castration-resistant prostate cancer: amulticenter retrospective analysis. Journal of Nuclear Medicine, 2016:p. jnumed. 116.173757) and PSMA I&T (Weineisen, M., et al., Developmentand first in human evaluation of PSMA I&T—A ligand for diagnosticimaging and endoradiotherapy of prostate cancer. Journal of NuclearMedicine, 2014. 55(supplement 1): p. 1083-1083, Eiber, M., et al.,Systemic radioligand therapy with 177Lu-PSMA I&T in patients withmetastatic castration-resistant prostate cancer. Journal of NuclearMedicine, 2016. 57(supplement 2): p. 61-61; Schottelius, M., et al.,[111 In] PSMA-I&T: expanding the spectrum of PSMA-I&T applicationstowards SPECT and radioguided surgery. EJNMMI research, 2015. 5(1):p. 1) are applied in clinical settings for palliative treatment ofprostate cancer patients. The chelating unit DOTA and the relatedDOTAGA, allow not only imaging but also therapeutical applications,since the scope for possible radiometal chelation encompasses ¹¹¹In,¹⁷⁷Lu, ⁹⁰Y and ²¹³Bi amongst others. [¹¹¹In] PSMA I&T was alreadyclinically implemented for radioguided surgery to assist the surgeonduring excision of the malignant tissue (Schottelius, M., et al., [111In] PSMA-I&T: expanding the spectrum of PSMA-I&T applications towardsSPECT and radioguided surgery. EJNMMI research, 2015. 5(1): p. 1).Likewise, the recent developed and clinically tested PSMA Inhibitor PSMAI&S (imaging and surgery) demonstrated highly encouraging results (Robu,S., et al., Preclinical evaluation and first patient application of99mTc-PSMA-I&S for SPECT imaging and radioguided surgery in prostatecancer. Journal of Nuclear Medicine, 2016: p. jnumed. 116.178939).

Endoradiotherapeutic approaches with [¹⁷⁷Lu]PSMA I&T demonstratedefficiency, tolerability and high safety potential in patients receivingup to four cycles with 7.4 GBq. The obtained dosimetric values for organradiation revealed, that especially the kidneys and the salivary glandsreceive the highest dose after tumor lesions. Similar radiation valueswere shown for PSMA DKFZ 617 and [¹⁸F]DCFPyL (Rowe, S. P., et al.,PSMA-Based [18F] DCFPyL PET/CT Is Superior to Conventional Imaging forLesion Detection in Patients with Metastatic Prostate Cancer. MolecularImaging and Biology, 2016: p. 1-9; Delker, A., et al., Dosimetry for177Lu-DKFZ-PSMA-617: a new radiopharmaceutical for the treatment ofmetastatic prostate cancer. European journal of nuclear medicine andmolecular imaging, 2016. 43(1): p. 42-51; Kabasakal, L., et al.,Pre-therapeutic dosimetry of normal organs and tissues of 177Lu-PSMA-617prostate-specific membrane antigen (PSMA) inhibitor in patients withcastration-resistant prostate cancer. European journal of nuclearmedicine and molecular imaging, 2015. 42(13): p. 1976-1983; Yadav, M.P., et al., 177Lu-DKFZ-PSMA-617 therapy in metastatic castrationresistant prostate cancer: safety, efficacy, and quality of lifeassessment. European Journal of Nuclear Medicine and Molecular Imaging,2016: p. 1-11). These elevated numbers are explainable by thephysiologic expression of PSMA (Silver, D. A., et al., Prostate-specificmembrane antigen expression in normal and malignant human tissues.Clinical Cancer Research, 1997. 3(1): p. 81-85) and the renal excretionof the radiolabeled compound. The occasional occurring renal andhematological toxicities after administration are usually reversible,although is legitimate concern about chronic toxicity especially inpatients with overall long survival rates, like in PCa patients.Therefore, a suitable concept is needed to reduce the unwanted radiationand simultaneously to increase the tumor uptake.

In view of the above, the technical problem underlying the presentinvention can be seen in the provision of means and methods ofalleviating radiation-induced side effects of PSMA-targetingradiolabeled diagnostics and therapeutics. A further technical problemcan be seen in the provision of means and methods to increase tumoruptake of such diagnostics and therapeutics. More generally speaking,the technical problem can be seen in the provision of improved PSMAbinding agents.

The technical problem is solved by the subject-matter summarized in theattached claims and explained in further detail below.

In particular, the present invention provides, in a first aspect, acompound of formula (I), or a pharmaceutically acceptable salt thereof,

wherein:

-   m is an integer of 2 to 6, preferably 2 to 4, more preferably 2;-   n is an integer of 2 to 6, preferably 2 to 4, more preferably 2 or    4;-   R^(1L) is CH₂, NH or O, preferably NH;-   R^(2L) is C or P(OH), preferably C;-   R^(3L) is CH₂, NH or O, preferably NH;-   X¹ is selected from an amide bond, an ether bond, a thioether bond,    an ester bond, a thioester bond, a urea bridge, and an amine bond,    and is preferably an amide bond;-   L¹ is a divalent linking group with a structure selected from an    oligoamide, an oligoether, an oligothioether, an oligoester, an    oligothioester, an oligourea, an oligo(ether-amide), an    oligo(thioether-amide), an oligo(ester-amide), an    oligo(thioester-amide), oligo(urea-amide), an    oligo(ether-thioether), an oligo(ether-ester), an    oligo(ether-thioester), an oligo(ether-urea), an    oligo(thioether-ester), an oligo(thioether-thioester), an    oligo(thioether-urea), an oligo(ester-thioester), an    oligo(ester-urea), and an oligo(thioester-urea), preferably with a    structure selected from an oligoamide and an oligo(ester-amide),-    which linking group may carry a group EDS;-   X² is selected from an amide bond, an ether bond, a thioether bond,    an ester bond, a thioester bond, a urea bridge, and an amine bond,    and is preferably an amide bond;-   R² is an optionally substituted aryl group or an optionally    substituted aralkyl group, which aryl group or aralkyl group may be    substituted on its aromatic ring with one or more substituents    selected from halogen, preferably I, and —OH;-   R³ is an optionally substituted aryl group or an optionally    substituted aralkyl group, which aryl group or aralkyl group may be    substituted on its aromatic ring with one or more substituents    selected from halogen, preferably I, and —OH;-   r is 0 or 1, preferably 1;-   p is 0 or 1;-   q is 0 or 1;-    and preferably p+q=1;-   R⁴ is selected from an aryl group and a group EDS;-   X³ is selected from an amide bond, an ether bond, a thioether bond,    an ester bond, a thioester bond, a urea bridge, an amine bond, and a    group of the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) andthe other marked bond attaches X³ to the remainder of the compound offormula (I);

-    and is preferably an amide bond;-   R^(M) is a marker group which comprises a chelating group optionally    containing a chelated non-radioactive or radioactive cation;

and wherein the group EDS is contained at least once in the compound offormula (I) and has a structure selected from (E-1A), (E-1B), (E-2A) and(E-2B):

wherein

marks the bond which attaches the group EDS, to the remainder of thecompound of formula (I);

s is 1, 2 or 3, preferably 1 or 2, and more preferably 1;

t is 1, 2 or 3, preferably 1 or 2, and more preferably 2;

R^(5A) is, independently for each occurrence for s>1, an electronwithdrawing substituent, which is preferably selected from —NO₂ and—COOH, and which is more preferably —COOH, and wherein the bond betweenR^(5A) and the phenyl ring indicates that the s groups R^(5A) replace shydrogen atoms at any position on the phenyl ring;

R^(5B) is, independently for each occurrence for s>1, a substituentcarrying an electron lone pair at the atom directly attached to thephenyl ring shown in formula (E-1B), which substituent is preferablyselected from —OH and —NH₂, and which is more preferably —NH₂, andwherein the bond between R^(5B) and the phenyl ring indicates that the sgroups R^(5B) replace s hydrogen atoms at any position on the phenylring;

R^(6A) is, independently for each occurrence for t>1, an electronwithdrawing substituent, which is preferably selected from —NO₂ and—COOH, and which is more preferably —COOH, and wherein the bond betweenR^(6A) and the phenyl ring indicates that the t groups R^(6A) replace thydrogen atoms at any position on the phenyl ring; and

R^(6B) is, independently for each occurrence for t>1, a substituentcarrying an electron lone pair at the atom directly attached to thephenyl ring shown in formula (E-1B), which substituent is preferablyselected from —OH and —NH₂, and which is more preferably —OH, andwherein the bond between R^(6B) and the phenyl ring indicates that the tgroups R^(6B) replace t hydrogen atoms at any position on the phenylring.

The introduction of an EDS substituent as shown above, wherein anaromatic ring carries one or more substituents with a high electrondensity selected from an electron withdrawing substituent and asubstituent carrying an electron lone pair leads to several unexpectedadvantages. These advantages include increased affinity, improvedinternalization, elevated tumor cell retention, lower unspecificbinding, reduced renal accumulation and an increased tumor uptake.

Especially the reduced unspecific uptake in organs other than prostateleads to less unwanted radiation and reduces radiation-induced sideeffects.

To exemplify these advantages, we refer to the properties of theparticularly preferred compounds herein designated PSMA-71 and PSMA-66which is discussed in more detail below.

In particular, nanomolar affinity (5.3±2.0 nM vs. 7.9±2.4 nM),drastically improved internalization (206.8±1.7% vs. 75.5±1.6%)demonstrate the superiority of [¹⁷⁷Lu]PSMA-71 when compared to[¹⁷⁷Lu]PSMA I&T. The biodistribution data revealed that [¹⁷⁷Lu]PSMA-71exhibited markedly higher tumor (14.29±0.89 vs. 4.06±1.12% ID/g,respectively) uptake compared to [¹⁷⁷Lu]PSMA I&T while the renalaccumulation was similar (32.36±2.49 vs. 34.66±17.20% ID/g,respectively).

Similarly, lower nanomolar affinity (3.8±0.3 nM vs. 7.9±2.4 nM),drastically improved internalization (297.8±2.0% vs. 75.5±1.6%),elevated in vitro tumor cell retention (90.1±3.5% vs. 62.8±0.4%, 60 minincubation) together with lower unspecific binding in vivo, reducedrenal accumulation (117.5±6.9% ID/g vs. 128.9±10.7% ID/g) and a morethan twofold increase in tumor uptake (10.0±0.4% vs. 4.7±1.0% ID/g),demonstrate the superiority of [¹⁷⁷Lu]PSMA-66 in direct comparison to[¹⁷⁷Lu]PSMA I&T.

As noted above, salts of the compounds of the invention includingcompounds of formula (I) (and including their preferred embodiments) arealso suitable for use in the context of the invention. It will beunderstood that these salts are generally pharmaceutically acceptablesalt forms of these compounds which may be formed, e.g., by protonationof an atom carrying an electron lone pair which is susceptible toprotonation, such as an amino group, with an inorganic or organic acid,or as a salt of a carboxylic acid group with a physiologicallyacceptable cation as they are well known in the art. Exemplary baseaddition salts comprise, for example, alkali metal salts such as sodiumor potassium salts; alkaline-earth metal salts such as calcium ormagnesium salts; ammonium salts; aliphatic amine salts such astrimethylamine, triethylamine, dicyclohexylamine, ethanolamine,diethanolamine, triethanolamine, procaine salts, meglumine salts,diethanol amine salts or ethylenediamine salts; aralkyl amine salts suchas N,N-dibenzylethylenediamine salts, benetamine salts; heterocyclicaromatic amine salts such as pyridine salts, picoline salts, quinolinesalts or isoquinoline salts; quaternary ammonium salts such astetramethylammonium salts, tetraethylammonium salts,benzyltrimethylammonium salts, benzyltriethylammonium salts,benzyltributylammonium salts, methyltrioctylammonium salts ortetrabutylammonium salts; and basic amino acid salts such as argininesalts or lysine salts. Exemplary acid addition salts comprise, forexample, mineral acid salts such as hydrochloride, hydrobromide,hydroiodide, sulfate salts, nitrate salts, phosphate salts (such as,e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts),carbonate salts, hydrogencarbonate salts or perchlorate salts; organicacid salts such as acetate, propionate, butyrate, pentanoate, hexanoate,heptanoate, octanoate, cyclopentanepropionate, undecanoate, lactate,maleate, oxalate, fumarate, tartrate, malate, citrate, nicotinate,benzoate, salicylate or ascorbate salts; sulfonate salts such asmethanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,benzenesulfonate, p-toluenesulfonate (tosylate), 2-naphthalenesulfonate,3-phenylsulfonate, or camphorsulfonate salts; and acidic amino acidsalts such as aspartate or glutamate salts.

Further examples of pharmaceutically acceptable salts include, but arenot limited to, acetate, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, butyrate, calcium edetate, camphorate, camphorsulfonate,camsylate, carbonate, chloride, citrate, clavulanate,cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate,edetate, edisylate, estolate, esylate, ethanesulfonate, formate,fumarate, gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate,mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like(see, for example, S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm.Sci., 66, pp. 1-19 (1977)).

It is understood that throughout the present specification the term“compound” encompasses solvates, polymorphs, prodrugs, codrugs,cocrystals, tautomers, racemates, enantiomers, or diastereomers ormixtures thereof unless mentioned otherwise.

When the compounds of the present invention are provided in crystallineform, the structure can contain solvent molecules. The solvents aretypically pharmaceutically acceptable solvents and include, amongothers, water (hydrates) or organic solvents. Examples of possiblesolvates include ethanolates and iso-propanolates.

The term “codrug” refers to two or more therapeutic compounds bonded viaa covalent chemical bond. A detailed definition can be found, e.g., inN. Das et al., European Journal of Pharmaceutical Sciences, 41, 2010,571-588.

The term “cocrystal” refers to a multiple component crystal in which allcomponents are solid under ambient conditions when in their pure form.These components co-exist as a stoichiometric or non-stoichiometricratio of a target molecule or ion (i.e., compound of the presentinvention) and one or more neutral molecular cocrystal formers. Adetailed discussion can be found, for example, in Ning Shan et al., DrugDiscovery Today, 13(9/10), 2008, 440-446 and in D. J. Good et al.,Cryst. Growth Des., 9(5), 2009, 2252-2264.

The compounds of the present invention can also be provided in the formof a prodrug, namely a compound which is metabolized in vivo to theactive metabolite. Suitable prodrugs are, for instance, esters. Specificexamples of suitable groups are given, among others, in US 2007/0072831in paragraphs [0082] to [0118] under the headings prodrugs andprotecting groups.

To the extent compounds of the invention exhibit a pH-dependent chargedstate, it is understood that all possible charged states are embraced. Apreferred pH range in this regard is from 0 to 14.

To the extent a compound according to the invention bears a net charge,it is understood that the compound is provided in electroneutral form.This is achieved by one or more counterions, preferred counterions beingdefined in relation to the term “salt” herein above.

In formula (I), m is an integer of 2 to 6. Preferably, m is 2 to 4, morepreferably 2. R^(1L) is CH₂, NH or O, preferably NH. R^(2L) is C orP(OH), preferably C. R^(3L) is CH₂, NH or O, preferably NH. Thus,compounds of formula (I) or their salts are also preferred wherein m is2, R^(1L) is NH, R^(2L) is C, and R^(3L) is NH.

n is an integer of 2 to 6, preferably 2 to 4, more preferably 2 or 4,and most preferably 2.

Thus, compounds of formula (I) or their salts are particularly preferredwherein m is 2, n is 2 or 4, R^(1L) is NH, R^(2L) is C, and R^(3L) isNH. More preferred are compounds of formula (I) or their salts wherein mis 2, n is 2, R^(1L) is NH, R^(2L) is C, and R^(3L) is NH.

X¹ in formula (I) is selected from an amide bond (i.e. —C(O)—NH—), anether bond (i.e. —O—), a thioether bond (i.e. —S—), an ester bond (i.e.—C(C)—C—, a thioester bond (i.e. —C(S)—C— or —C(O)—S—), a urea bridge(i.e. —NH—C(O)—NH—), and an amine bond (i.e. —NH—). Preferred as X¹ isthe amide bond.

Moreover, it is further preferred in formula (I) that either n is 2 andX¹ is the amide bond with the carbon atom of the amide bond —C(O)—NH—being attached to the group —(CH₂)_(n)—, or that n is 4 and X¹ is theamide bond with the carbon atom of the amide bond —C(O)—NH— beingattached to the group —(CH₂)_(n)—. Among these, more preferred is theoption that n is 2 and X¹ is the amide bond with the carbon atom of theamide bond —C(O)—NH— being attached to the group —(CH₂)_(n)—.

Thus, particularly preferred are compounds of formula (I) and theirsalts wherein, in formula (I), m is 2, n is 2, R^(1L) is NH, R^(2L) isC, R^(3L) is NH, and X¹ is an amide bond with the carbon atom of theamide bond —C(O)—NH— being attached to the group —(CH₂)_(n)—.

L¹ in formula (I) is a divalent linking group with a structure selectedfrom an oligoamide, an oligoether, an oligothioether, an oligoester, anoligothioester, an oligourea, an oligo(ether-amide), anoligo(thioether-amide), an oligo(ester-amide), anoligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), anoligo(ether-ester), an oligo(ether-thioester), an oligo(ether-urea), anoligo(thioether-ester), an oligo(thioether-thioester), anoligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea),and an oligo(thioester-urea), preferably with a structure selected froman oligoamide and an oligo(ester-amide), and more preferably with anoligoamide structure, which linking group may carry a group EDS.

The term “oligo” as used in the definition of L¹ in the termsoligoamide, oligoether, oligothioether, oligoester, oligothioester,oligourea, oligo(ether-amide), oligo(thioether-amide),oligo(ester-amide), oligo(thioester-amide), oligo(urea-amide),oligo(ether-thioether), oligo(ether-ester), oligo(ether-thioester),oligo (ether-urea), oligo(thioether-ester), oligo(thioether-thioester),oligo(thioether-urea), oligo(ester-thioester), oligo(ester-urea), andoligo(thioester-urea) is preferably to be understood as referring to agroup wherein 2 to 20, more preferably wherein 2 to 10 subunits arelinked by the type of bonds specified in the same terms. As will beunderstood by the skilled reader, where two different types of bonds areindicated in brackets, both types of bonds are contained in theconcerned group (e.g. in “oligo (ester-amide)”, ester bonds and amidebonds are contained).

It is more preferred that L¹ has a structure selected from an oligoamidewhich comprises a total of 1 to 5, more preferably a total of 1 to 3,and most preferably a total of 1 or 2 amide bonds within its backbone,and an oligo(ester-amide) which comprises a total of 2 to 5, morepreferably a total of 2 to 3, and most preferably a total of 2 amide andester bonds within its backbone. In a particularly preferred embodiment,L¹ represents a divalent linking group with an oligoamide structurewhich comprises 1 or 2 amide bonds within its backbone.

Furthermore, L¹ may carry a group EDS (i.e. a group carrying asubstituent with a high electron density or “electron densesubstituent”) as defined herein, i.e. a group EDS which is covalentlyattached to L¹. Preferably, the optional group EDS is attached as asubstituent to the backbone of the divalent linking group L¹, L¹ havinga structure as defined above selected from an oligoamide, an oligoether,an oligothioether, an oligoester, an oligothioester, an oligourea, anoligo(ether-amide), an oligo(thioether-amide), an oligo(ester-amide), anoligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), anoligo(ether-ester), an oligo(ether-thioester), an oligo(ether-urea), anoligo(thioether-ester), an oligo(thioether-thioester), anoligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea),and an oligo(thioester-urea), preferably a structure selected from anoligoamide and an oligo(ester-amide), and most preferably an oligoamidestructure, which backbone extends between X¹ and X² in the compound offormula (I). Also in this regard, the further preferred definitions forL¹ apply, i.e. it is more preferred that L¹ has a structure selectedfrom an oligoamide which comprises a total of 1 to 5, more preferably atotal of 1 to 3, and most preferably a total of 1 or 2 amide bondswithin its backbone, and an oligo(ester-amide) which comprises a totalof 2 to 5, more preferably a total of 2 to 3, and most preferably atotal of 2 amide and ester bonds within its backbone. In a particularlypreferred embodiment, L¹ represents a divalent linking group with anoligoamide structure which comprises 1 or 2 amide bonds within itsbackbone.

In accordance with the above, L¹ may carry one or more, e.g. 2 or 3,groups EDS. However, it is preferred that L¹ does not carry a group EDS,or that L¹ carries one group EDS, and it is more preferred that L¹carries one group EDS.

If L¹ carries a group EDS (including the more preferred case that EDScarries one group EDS), it is preferred that the group EDS has astructure selected from (E-1A), (E-2A) and (E-2B). It is more preferredthat the group EDS has a structure selected from (E-2A) and (E-2B), andmost preferably it has the structure (E-2A).

As will be understood by the skilled reader, the indication that L¹ maycarry a group EDS serves as information on a possible position of thisgroup in the compounds in accordance with the invention. The fact thatL¹ may carry a group EDS does not impose a restriction on the presenceof other groups that may be present e.g. as alternative or additionalsubstituents on the backbone of L¹. For example, it is preferred thatthe linking group L¹ contains one or more, such as two, groups which areindependently selected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and—NHC(NH)NH₂ attached as substitutents to its backbone. More preferably,the linking group L¹ contains one or more, such as two, groups —COOH,attached as substitutents to its backbone.

In formula (I), X² is selected from an amide bond, an ether bond, athioether bond, an ester bond, a thioester bond, a urea bridge, and anamine bond, and is preferably an amide bond.

It is more preferred that the nitrogen atom of the amide bond —C(O)—NH—is attached to L¹.

Thus, it is also preferred that X¹ and X² are both amide bonds,especially amide bonds arranged in the preferred orientations furtherdefined above.

In line with the above, it is preferred that the moiety —X²-L¹-X¹— informula (I) has a structure selected from:

*—C(O)—NH—R⁷—NH—C(O)—R⁸—C(O)—NH—  (L-1),

*—C(O)—NH—R^(9A)—NH—C(O)—R^(10A)—C(O)—NH—R^(11A)—NH—C(O)—  (L-2A), and

*—C(O)—NH—R^(9B)—C(O)—NH—R^(10B)—C(O)—NH—R^(11B)—NH—C(O)—  (L-2B);

wherein the amide bond marked with * is attached to the carbon atomcarrying R² in formula (I), and wherein

R⁷, R⁸, R^(9A), R^(9B), R^(11A) and R^(11B) are independently selectedfrom optionally substituted C2 to C10 alkanediyl, preferably optionallysubstituted linear C2 to C10 alkanediyl, which alkanediyl groups mayeach be substituted by one or more substitutents independently selectedfrom —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, —NHC(NH)NH₂, and a group EDS, andR^(10A) and R^(10B) are selected from optionally substituted C2 to C10alkanediyl, preferably optionally substituted linear C2 to C10alkanediyl, and optionally substituted C6 to C10 arenediyl, preferablyphenylene, which alkanediyl and arenediyl group may each be substitutedby one or more substitutents independently selected from —OH, —OCH₃,—COOH, —COOCH₃, —NH₂, —NHC(NH)NH₂, and a group EDS. R^(10A) ispreferably optionally substituted C2 to C10 alkanediyl, more preferablyoptionally substituted linear C2 to C10 alkanediyl as defined above.R^(10B) is preferably optionally substituted C6 to C10 arenediyl asdefined above, more preferably a phenylene group, e.g. para-phenylenegroup.

In the groups of formula (L-1), (L-2A) and (L-2B), it is preferred thatthe optional substituent on R⁷ is —COOH, that the optional substituentof R⁸ is a group EDS, that the optional substituent on R^(9A) and R^(9B)is —COOH, that the optional substituent on R^(10A) is a group EDS, andthat the optional substituent on R_(11A) and R^(11B) is —COOH.

It is also preferred that each of the groups of formula (L-1) and (L-2A)carries a group EDS as at least one substituent, as explained abovepreferably as a substituent of R⁸ and R^(10A). Also in this context, itis preferred that the group EDS has a structure selected from (E-1A),(E-2A) and (E-2B). It is more preferred that the group EDS has astructure selected from (E-2A) and (E-2B), and most preferably it hasthe structure (E-2A).

Furthermore, it is preferred that the total number of carbon atoms in R⁷and R⁸ of formula (L-1) is 6 to 20, more preferably 6 to 16, withoutcarbon atoms contained in optional substituents, that the total numberof carbon atoms in R^(9A), R^(10A) and R^(11A) of formula (L-2A) is 6 to20, more preferably 6 to 16, without carbon atoms contained in optionalsubstituents, and that the total number of carbon atoms in R^(9B),R^(10B) and R^(11B) of formula (L-2B) is 6 to 20, more preferably 6 to16, without carbon atoms contained in optional substituents.

It will be understood from the information provided with respect topreferred meanings of n and X¹ above that it is still further preferredthat —X²-L¹-X¹— in formula (I) has a structure (L-1) if n is 4, and that—X²-L¹-X¹— in formula (I) has a structure (L-2A) or (L-2B) if n is 2.

In line with the above definitions, it is even further preferred thatthe moiety —X²-L¹-X¹— in formula (I) has a structure selected from:

*—C(O)—NH—CH(COOH)—R¹²—NH—C(O)—R¹³—C(O)—NH—  (L-3),

*—C(O)—NH—CH(COOH)—R¹⁴—NH—C(O)—R¹⁵—C(O)—NH—R¹⁶—CH(COOH)—NH—C(O)—  (L-4), and

*—C(O)—NH—CH(COOH)—R¹⁷—C(O)—NH—R¹⁸—C(O)—NH—R¹⁹—CH(COOH)—NH—C(O)—  (L-5);

wherein the bond marked with * is attached to the carbon atom carryingR² in formula (I),

R¹² and R¹⁴ are independently selected from linear C2 to C6 alkanediyl,preferably from linear C3 to C6 alkanediyl,

R¹³ is a linear C2 to C10 alkanediyl, preferably a linear C4 to C8alkanediyl,

R¹⁵ and R¹⁶ are independently selected from linear C2 to C6 alkanediyl,preferably from linear C2 to C4 alkanediyl,

and wherein each of R¹³ and R¹⁵ may carry one group EDS as a substituentand preferably each of R¹³ and R¹⁵ carries one group EDS as asubstituent,

R¹⁷ is a linear C2 to C6 alkanediyl, preferably a linear C2 to C4alkanediyl,

R¹⁸ is a phenylene group, e.g. a para-phenylene group, and

R¹⁹ is a linear C2 to C6 alkanediyl, preferably a linear C2 to C4alkanediyl.

Also in this context, it is preferred that the group EDS which may beattached to R¹³ and R¹⁵ has a structure selected from (E-1A), (E-2A) and(E-2B). It is more preferred that the group EDS has a structure selectedfrom (E-2A) and (E-2B), and most preferably it has the structure (E-2A).

Furthermore, it is preferred that the total number of carbon atoms inR¹² and R¹³ in formula (L-3), without carbon atoms contained in thegroup EDS as substituent, is 6 to 16, more preferably 6 to 14, and thetotal number of carbon atoms in R¹⁴, R¹⁵ and R¹⁶ in formula (L-4),without carbon atoms contained in the group EDS as substituent, is 6 to16, more preferably 6 to 14.

It will be understood from the information provided with respect topreferred meanings of n and X¹ above that it is particularly preferredthat —X²-L¹-X¹— in formula (I) has a structure (L-3) if n is 4, and that—X²-L¹-X¹— in formula (I) has a structure (L-4) or (L-5) if n is 2.

It is specifically preferred that, in formula (I), n is 2 and the moiety—X²-L¹-X¹— has one of the following structures:

*—C(O)—NH—CH(COOH)—(CH₂)₄—NH—C(O)—CH(EDS)—CH₂—C(O)—NH—(CH₂)₃—CH(COOH)—NH—C(O)—  (L-6)

*—C(O)—NH—CH(COOH)—(CH₂)₂—C(O)—NH-Ph-C(O)—NH—(CH₂)₃—CH(COOH)—NH—C(O)—  (L-7)

wherein the bond marked with * is attached to the carbon atom carryingR² in formula (I), EDS is a group EDS as defined herein, including itspreferred embodiments, and Ph is a para-phenylene group.

Also in this context, it is preferred that the group EDS has a structureselected from (E-1A), (E-2A) and (E-2B). It is more preferred that thegroup EDS has a structure selected from (E-2A) and (E-2B), and mostpreferably it has the structure (E-2A).

In formula (I), R² is an optionally substituted aryl group or anoptionally substituted aralkyl group, preferably an optionallysubstituted aralkyl group. As will be understood, the term “aralkylgroup” as used herein refers to an alkyl group wherein a hydrogen atomis replaced by an aryl group as a substituent. Preferably, the aralkylgroup is a group wherein one aryl group is bound to an alkanediyl group.The aryl group or aralkyl group represented by R² may be substituted onits aromatic ring with one or more substituents selected from halogen,preferably I, and —OH. The aryl and the aryl portion of the aralkylgroup are preferably selected from phenyl and naphthyl, such as2-naphthyl. The alkanediyl portion of the aralkyl group is preferably aC1-C4 alkanediyl group, more preferably a —CH₂— group. Thus, R² is morepreferably selected from optionally substituted —CH₂-phenyl, andoptionally substituted —CH₂-naphtyl, in particular optionallysubstituted —CH₂-(2-naphtyl). Optionally substituted —CH₂-(2-naphtyl) isa particularly preferred option for R².

The optionally substituted aryl group and the aryl portion of theoptionally substituted aralkyl group, including their preferredembodiments, may be substituted with one or more substituents selectedfrom halogen, preferably I, and —OH. Thus, one or more than one, e.g. 2or 3, substituent(s) selected from halogen, preferably I, and —OH can bepresent. However, it is preferred that R² is non-substituted.

In view of the above, it will be understood that, R² is most preferablynon-substituted —CH₂-(naphtyl), and that the naphthyl group is mostpreferably a 2-naphtyl group to provide R² as —CH₂-(2-naphthyl).

In formula (I), R³ is an optionally substituted aryl group or anoptionally substituted aralkyl group, preferably an optionallysubstituted aralkyl group. The aryl group or aralkyl group may besubstituted on its aromatic ring with one or more substituents selectedfrom halogen, preferably I, and —OH. The aryl and the aryl portion ofthe aralkyl group are preferably selected from phenyl and naphthyl, suchas 2-naphthyl. It is more preferred that the aryl and the aryl portionof the aralkyl are phenyl. The alkanediyl portion of the aralkyl groupis preferably a C1-C4 alkanediyl group, more preferably a —CH₂— group.Thus, R³ is more preferably optionally substituted —CH₂-phenyl.

The optionally substituted aryl group and the aryl portion of theoptionally substituted aralkyl group, including their preferredembodiments, may be substituted with one or more substituents selectedfrom halogen, preferably I, and —OH. Thus, one or more than one, e.g. 2or 3, substituent(s) selected from halogen, preferably I, and —OH can bepresent.

Preferably, R³ is substituted with one substituent which is —OH, or witha combination of one substituent —OH and one substituent —I.

Thus, it is particularly preferred that R³ is —CH₂-phenyl substituted onthe phenyl ring with one substituent which is —OH, or with a combinationof one substituent —OH and one substituent —I, and it is most preferredthat the substituent —OH is present in the para-position of the phenylring relative to the —CH₂— group.

In line with the above, it is preferred in formula (I) that R² is agroup of the formula

and R³ is a group of the formula

wherein

marks the bond which attaches R² and R³, respectively, to the remainderof the compound of formula (I).

Even more strongly preferred is the combination of R² and R³ wherein R²is a group of the formula

and R³ is a group of the formula

wherein

marks the bond which attaches R² and R³, respectively, to the remainderof the compound.

In formula (I), r can be 0 or 1, and it is preferred that r is 1.

Furthermore, as explained above, p is 0 or 1 and q is 0 or 1, and it ispreferred that p+q=1. More preferably, p is 0 and q is 1.

R⁴ in formula (I) is selected from an aryl group and a group EDS. Thearyl is preferably selected from phenyl and naphthyl, such as2-naphthyl. Thus, R⁴ is more preferably selected from phenyl, naphtyl,such as 2-naphthyl, and a group EDS. It is most preferably a group EDS.

If R⁴ is a group EDS, it is preferred that the group EDS has a structureselected from (E-1A), (E-2A) and (E-1B).

X³ is selected from an amide bond, an ether bond, a thioether bond, anester bond, a thioester bond, a urea bridge, an amine bond, and a groupof the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) andthe other marked bond attaches X³ to the remainder of the molecule.

Preferably, X³ is selected from an amide bond and a group of the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) andthe other marked bond attaches X³ to the remainder of the molecule.

In a more preferred embodiment, X³ is an amide bond —C(O)—NH— with thecarbon atom attached to R^(M).

R^(M) is a marker group which comprises a chelating group optionallycontaining a chelated non-radioactive or radioactive cation.

As will be understood by the skilled reader, the above definitionaccording to which R^(M) comprises a chelating group encompasses thecase that R^(M) is a chelating group; in this case the chelating groupis typically directly bound to X³;

and the case that R^(M) comprises, together with the chelating group,e.g. a further linker moiety; in this case the chelating group can beindirectly bound via this further linker moiety to X³.

The chelating group provided by R^(M) is suitable to form a chelate witha radioactive or non-radioactive cation. Suitable chelating groups fordiverse cations are well known in the art, and can be used in thecontext of the present invention.

The chelating group optionally containing a chelated non-radioactive orradioactive cation is preferably selected from a chelating groupcomprising at least one of

-   (i) a macrocyclic ring structure with 8 to 20 ring atoms of which 2    or more, preferably 3 or more, are selected from oxygen atoms,    sulfur atoms and nitrogen atoms; and-   (ii) an acyclic, open chain chelating structure with 8 to 20 main    chain atoms of which 2 or more, preferably 3 or more are heteroatoms    selected from oxygen atoms, sulfur atoms and nitrogen atoms.

An exemplary chelating group, and thus also an exemplary group R^(M), isa residue of a chelating agent selected frombis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a),cyclohexyl-1,2-diaminetetraacetic acid (CDTA),4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA),N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide(DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan(DO2A) 1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid(DOTA), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-triaceticacid]-pentanedioic acid (DOTAGA),—N,N′-dipyridoxylethylendiamine-N,N′-diacetate-5,5′-bis(phosphat)(DPDP), diethylenetriaminepentaacetic acid (DTPA),ethylenediamine-N,N′-tetraacetic acid (EDTA),ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED),hydroxyethyldiaminetriacetic acid (HEDTA),1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate(HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC),1,4,7-triazacyclononan-1-succinic acid-4,7-diacetic acid (NODASA),1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane(NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane(TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA),terpyridin-bis(methyleneamintetraacetic acid (TMT),1,4,7,10-tetraazacyclotridecan-N,N′,N″,N′″-tetraacetic acid (TRITA),triethylenetetraaminehexaacetic acid (TTHA),N,N′-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6 (H₂macropa)and4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}heptanedioic acidbis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide](THP);

which residue is provided by covalently binding a carboxyl groupcontained in the chelating agent to the remainder of the compound via anester or an amide bond, preferably an amide bond. It will be understoodby the skilled reader that, in formula (I), this ester or amide bond canin this case be encompassed by X³ or can preferably be represented byX³.

Among these chelating agents, DOTA and DOTAGA are preferred.

Thus, it is also preferred that R^(M)—X³— in formula (I) is a group ofthe formula

wherein the bond marked with

is attached to the remainder of the compound of formula (I), and whereinthe chelating group may contain a chelated non-radioactive orradioactive cation.

Exemplary radioactive cations that are optionally chelated by thechelating group are selected from the cations of ⁴⁴Sc, ⁴⁷Sc, ⁵¹Cr,^(52m)Mn, ⁵⁸Co, ⁵²Fe, ⁵⁶Ni, ⁵⁷Ni, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁸Ga, ⁶⁷Ga,⁸⁹Zr, ⁹⁰Y, ⁸⁹Y, ^(94m)Tc, ^(99m)Tc, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag,^(110m)In, ¹¹¹In, ^(113m)In, ^(114m)In, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr,¹⁴³Pr, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁹Er,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷²Tm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹¹Pt, ¹⁹⁷Hg, ¹⁹⁸Au, ¹⁹⁹Au,²¹²Pb, ²⁰³Pb, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, and ²²⁷Th, or acationic molecule comprising ¹⁸F, such as ¹⁸F-[AIF]²⁺.

Preferred chelated cations are selected from the cations of ⁴⁴Sc, ⁴⁷Sc,⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁹⁰Y, ¹¹¹In, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi,²¹³Bi, ²²⁵Ac, and ²²⁷Th, or a cationic molecule comprising ¹⁸F.

In formula (I), the group EDS is contained at least once, such that e.g.one, two or three groups EDS may be contained. Preferably, the compoundsor salts in accordance with the invention contain one group or twogroups EDS. As explained above, the group(s) EDS may be carried by L¹,and/or may be represented by R⁴.

The most preferred compounds of formula (I) or their salts are thosewhich contain one group EDS which is carried by the linking group L¹,including its preferred embodiments as set forth above, and those whichcontain two groups EDS, one being represented by R⁴ (i.e. r is 1) andone being carried by L¹, including its preferred embodiments as setforth above.

As set out above, the group EDS has a structure selected from (E-1A),(E-1B), (E-2A) and (E-2B):

wherein

marks the bond which attaches the group EDS, to the remainder of thecompound of formula (I);

s is 1, 2 or 3, preferably 1 or 2, and more preferably 1;

t is 1, 2 or 3, preferably 1 or 2, and more preferably 2;

R^(5A) is, independently for each occurrence for s>1, an electronwithdrawing substituent, which is preferably selected from —NO₂ and—COH, and which is more preferably —COOH, and wherein the bond betweenR^(5A) and the phenyl ring indicates that the s groups R^(5A) replace shydrogen atoms at any position on the phenyl ring;

R^(5B) is, independently for each occurrence for s>1, a substituentcarrying an electron lone pair at the atom directly attached to thephenyl ring shown in formula (E-1B), which substituent is preferablyselected from —OH and —NH₂, and which is more preferably —NH₂, andwherein the bond between R^(5B) and the phenyl ring indicates that the sgroups R^(5B) replace s hydrogen atoms at any position on the phenylring;

R^(6A) is, independently for each occurrence for t>1, an electronwithdrawing substituent, which is preferably selected from —NO₂ and—COOH, and which is more preferably —COOH, and wherein the bond betweenR^(6A) and the phenyl ring indicates that the t groups R^(6A) replace thydrogen atoms at any position on the phenyl ring; and

R^(6B) is, independently for each occurrence for t>1, a substituentcarrying an electron lone pair at the atom directly attached to thephenyl ring shown in formula (E-1B), which substituent is preferablyselected from —OH and —NH₂, and which is more preferably —OH, andwherein the bond between R^(6B) and the phenyl ring indicates that the tgroups R^(6B) replace t hydrogen atoms at any position on the phenylring.

It is generally preferred that, in the group EDS (E-1A), thesubstituents R^(5A) are the same for s>1 and are selected from —NO₂ and—COOH, and are more preferably —COOH, and that, in the group EDS (E-2A),the substituents R^(6A) are the same for t>1 and are selected from —NO₂and —COOH, and are more preferably —COOH.

It is likewise generally preferred that, in the group EDS (E-1B), thesubstituents R^(5B) are the same for s>1 and are selected from —OH and—NH₂, and are more preferably —NH₂, and that, in the group EDS (E-2B),the substituents R^(6B) are the same for t>1 and are selected from —OHand —NH₂, and are more preferably —OH.

Thus, it is further preferred that the compound of formula (I) containsa group EDS which has the formula (E-2A):

wherein

marks the bond which attaches the group EDS to the remainder of thecompound of formula (I); and

t is 1 or 2, and R^(6A) is —NO₂ or —COOH.

Most preferred as group EDS in the context of the present invention isthe group

In line with the above, preferred compounds of formula (I) areillustrated by the following formula (Ia)

wherein n, X¹, L¹, X², R², R³, R⁴, q, p, X³ and R^(M) are defined asabove, including their preferred embodiments, and wherein the group EDSis contained at least once and has a structure as defined above,including the preferred embodiments.

More preferred compounds of formula (I) are illustrated by the followingformula (Ib)

wherein n, X¹, L¹, X², R², R³, R⁴, X³ and R^(M) are defined as above,including their preferred embodiments, and wherein the group EDS iscontained at least once and has a structure as defined above, includingthe preferred embodiments.

Still more preferred compounds of formula (I) are illustrated by thefollowing formula (Ic)

wherein n, X¹, L¹, X², R⁴, X³ and R^(M) are defined as above, includingtheir preferred embodiments, and wherein the group EDS is contained atleast once and has a structure as defined above, including its preferredembodiments.

Still more preferred compounds of formula (I) are illustrated by thefollowing formulae (Id) and (Ie):

wherein R^(9A), R^(10A), R^(11A), R⁴, X³ and R^(M) are defined as above,including their preferred embodiments, and wherein either (i) R4 is agroup EDS with a structure as defined above, including its preferredembodiments, or (ii) R^(10A) carries one group EDS with a structure asdefined above, including its preferred embodiments, or both (i) and (ii)apply;

wherein R^(9B), R^(10B), R^(11B), R⁴, X³ and R^(M) are defined as above,including their preferred embodiments, and wherein R⁴ is a group EDSwith a structure as defined above, including its preferred embodiments.

Particularly preferred compounds of formula (I) are illustrated by thefollowing formulae (If) and (Ig)

wherein R^(9A), R^(10A), R^(11A), R⁴, X³ and R^(M) are defined as above,including their preferred embodiments, and wherein either (i) R4 is agroup EDS with a structure as defined above, including its preferredembodiments, or (ii) R^(10A) carries one group EDS with a structure asdefined above, including its preferred embodiments, or both (i) and (ii)apply;

wherein R^(9B), R^(10B), R^(11B), R⁴, X³ and R^(M) are defined as above,including their preferred embodiments, and wherein R⁴ is a group EDSwith a structure as defined above, including its preferred embodiments.

In a preferred embodiment, said chelating group has a radionuclide boundwhich radionuclide emits α-radiation. Radionuclides emitting α-radiationinclude ²¹²Bi, ²¹³Bi, and ²²⁵Ac.

As noted above, the introduction of the electron deficient substituentsdrastically increased the internalization capacities. This featureresults in higher tumor uptake and especially longer retention in thetumor tissue as demonstrated by the in vitro experiments (see examples).Since the complex of the chelator and alpha-particle emittingradionuclide is prone to decomplexation via physical recoil effect, thefeature of elongated intracellular retention will reduce the probabilityof freely circulating radionuclides in vivo and thus increase safety andreduce unwanted radiation.

Particularly preferred compounds of the invention are the following:

DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) (PSMA-36):

DOTAGA-F(4-NH₂)y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-49):

DOTAGA-F(4-NO₂)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-52):

2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-53):

DOTAGA-F(4-NH₂)y-2-naI-e(Abz-N⁵-orn-C⁴-EuE) (PSMA-60):

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-2,4-DNBA) (PSMA-61):

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-62):

2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N⁵-orn-C⁴-EuE) (PSMA-65):

DOTAGA-Dap(TMA)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-66):

DOTAGA-2-NaI-y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-71):

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-3,5-DHBA) (PSMA-78):

The advantageous properties of these inventive compounds can be seenfrom the data in Tables 1 and 2 below, which data is presentedgraphically in FIG. 1 .

TABLE 1 Summary of all in vitro investigated parameter for the EuK-basedPSMA inhibitors. E designates Glu, u urea and K designates Lys. Lowercase single letters (such as “y”) designate D-forms of the respectiveamino acid. The half maximal inhibitory concentration (IC₅₀) of the PSMAinhibitors was determined in a competitive binding assay using LNCaPcell (1.5 * 10⁵ cells/well, 1 h, 4° C., HBSS + 1% BSA) and([¹²⁵I]I-BA)KuE as radioligand. Internalized activity expressed in [%]as relative cellular uptake to ([¹²⁵I]I-BA)KuE (1.25 * 10⁵ cells/well,PLL-coated plates, c = 0.2 nM for ([¹²⁵I]I-BA)KuE and c = 1.0 nM for¹⁷⁷Lu-labeled PSMA inhibitors. DMEM/F-12 + 5% BSA, 37° C., 60 min). Dataare corrected for non-specific binding (10 μM 2-PMPA). IC₅₀ andinternalization data are expressed as mean ± SD (n = 3). Lipophilicityexpressed as logP (distribution coefficient in n-octanol/PBS) ofradiolabeled PSMA inhibitors. Data for logP expressed as mean ± SD (n =6). Albumin binding (HSA) expressed in [%] after logarithmic plottingand calibration (n = 1). Configuration describes simplified the N- toC-terminal structural composition of the peptide spacer and linkerwithout the chelator. IC₅₀ Internalization HSA PSMA inhibitorConfiguration [nM] [%] logP [%] [^(nat/177)Lu]PSMA I&T -y(3-I)fk- 7.9 ±2.4 75.5 ± 1.6 −4.12 ± 0.11 78.6 [^(nat/177)Lu]PSMA-617 3.8 ± 1.7 160.1± 1.5  n.d. 74.7 [^(nat/177)Lu]PSMA-36 -y(3-I)fk- ∥ -2,4-DNBA- 5.3 ± 1.0189.8 ± 37.5 n.d. 82.5 n.d. = not determined. “-II-” indicates asimplified conjugation.

TABLE 2 Summary of all in vitro investigated parameter for the EuE-basedPSMA inhibitors. The half maximal inhibitory concentration (IC₅₀) of thePSMA inhibitors was determined in a competitive binding assay usingLNCaP cell (1.5 * 10⁵ cells/well, 1 h, 4° C., HBSS + 1% BSA) and([¹²⁵I]I-BA)KuE as radioligand. Internalized activity expressed in [%]as relative cellular uptake to ([¹²⁵I]I-BA)KuE (1.25 * 10⁵ cells/well,PLL-coated plates, c = 0.2 nM for ([¹²⁵I]I- BA)KuE and c = 1.0 nM for¹⁷⁷Lu-labeled PSMA inhibitors. DMEM/F-12 + 5% BSA, 37° C., 60 min). Dataare corrected for non-specific binding (10 μM 2-PMPA). IC₅₀ andinternalization data are expressed as mean ± SD (n = 3). Lipophilicityexpressed as logP (distribution coefficient in n-octanol/PBS) ofradiolabeled PSMA inhibitors. Data for logP expressed as mean ± SD (n =6). Albumin binding (HSA) expressed in [%] after logarithmic plottingand calibration (n = 1). Configuration describes simplified the N- toC-terminal structural composition of the peptide spacer and linkerwithout the chelator. IC₅₀ Internalization HSA PSMA inhibitorConfiguration [nM] [%] logP [%] [^(nat/177)Lu]PSMA I&T -y(3-I)fk- ∥ -KuE7.9 ± 2.4  75.5 ± 1.6 −4.12 ± 0.11 78.6 [^(nat/177)Lu]PSMA-46-y-2-nal-k- ∥ -EuE 3.2 ± 1.1 216.2 ± 9.2 −4.21 ± 0.08 57.7[^(nat/177)Lu]PSMA-617 3.8 ± 1.7 160.1 ± 1.5 n.d. 74.7[^(nat/177)Lu]PSMA-52 -F(4-NO₂)-y-2-nal- 3.4 ± 0.2 229.9 ± 8.0 −4.11 ±0.07 95.4 k(Suc-N⁵-orn-C⁴- EuE) ^([nat/177)Lu]PSMA-53 2,4-DNBA- 3.2 ±0.5  293.6 ± 10.0 −4.08 ± 0.04 95.9 Dap(DOTAGA)-y-2-nal-k(Suc-N⁵-orn-C⁴- EuE) [^(nat/177)Lu]PSMA-61 -F(4-NH₂)y-2-nal- 4.5 ±0.4  359.5 ± 22.6 −4.07 ± 0.05 63.3 k(d[N⁵-orn-C⁴-EuE]- 2,4-DNBA)[^(nat/177)Lu]PSMA-62 -F(4-NH₂)y-2-nal- 4.0 ± 0.2 343.9 ± 6.0 −4.12 ±0.05 >91.0 k(d[N⁵-orn-C⁴-EuE]- TMA) [^(nat/177)Lu]PSMA-66-Dap(TMA)y-2-nal- 3.8 ± 0.3 297.8 ± 2.0 −4.25 ± 0.14 64.4k(d[N⁵-orn-C⁴-EuE]- TMA) [^(nat/177)Lu]PSMA-71 -2-Nal-y-2-nal-k(d[N⁵-5.3 ± 2.0 206.8 ± 1.7 −4.13 ± 0.09 98.2 orn-C⁴-EuE]-TMA)[^(nat/177)Lu]PSMA-49 -F(4-NH₂)y-2-nal- 2.5 ± 0.6 245.0 ± 4.2  −4.01 ±0.11. 74.2. k(Suc-N⁵-orn-C⁴-EuE) [^(nat/177)Lu]PSMA-60 -F(4-NH₂)y-2-nal-6.6 ± 1.5 267.4 ± 7.9 −3.85 ± 0.13 98.5 n.d. e(Abz-N⁵-orn-C⁴- n.d. EuE)[^(nat/177)Lu]PSMA-78 -F(4-NH₂)y-2-nal-  3.9 ± 0.6. 289.0 ± 6.2 n.d.n.d. k(d[N⁵-orn-C⁴-EuE]- 3,5-DHBA) [^(nat/177)Lu]PSMA-65 2,4-DNBA-   3.5 ± 0.3n.d.  340.2 ± 19.9    −4.15 ± 0.08n.d. 98.7 n.d.Dap(DOTAGA)y-2- nal-e(Abz-N⁵-orn-C⁴- EuE) n.d. = not determined. “-II-”indicates a simplified conjugation.

Preferred labeling schemes for these most preferred compounds are asdefined herein above.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising or consisting of one or more compounds or saltsof the invention as disclosed herein above.

In a further aspect, the present invention provides a diagnosticcomposition comprising or consisting of one or more compounds or saltsof the invention as disclosed herein above.

In a further aspect, the present invention provides a therapeuticcomposition comprising or consisting of one or more compounds or saltsof the invention as disclosed herein above.

The pharmaceutical composition may further comprise pharmaceuticallyacceptable carriers, excipients and/or diluents. Examples of suitablepharmaceutical carriers, excipients and/or diluents are well known inthe art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical, intradermal, intranasal or intrabronchialadministration. It is particularly preferred that said administration iscarried out by injection and/or delivery, e.g., to a site in thepancreas or into a brain artery or directly into brain tissue. Thecompositions may also be administered directly to the target site, e.g.,by biolistic delivery to an external or internal target site, like thepancreas or brain. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently.Pharmaceutically active matter may be present in amounts between 0.1 ngand 10 mg/kg body weight per dose; however, doses below or above thisexemplary range are envisioned, especially considering theaforementioned factors.

To the extent the above disclosed pharmaceutical composition, diagnosticcomposition and therapeutic composition comprises one or more compoundsof the invention, it is preferred that no further pharmaceuticallyactive compounds, diagnostically active compounds or therapeuticallyactive compounds are present. In the alternative, furthertherapeutically active, diagnostically active or pharmaceutically activecompounds may be present, for example, anticancer agents.

Combination of a therapeutic treatment with the compounds of the presentinvention might have a synergistic or cumulative treatment effect,similar to the treatment of neuroendocrine tumors with [¹¹⁷Lu]DOTATATEradiotherapy in combination with chemotherapy or immunotherapies. Afirst phase 3 study comparing the combination of ¹⁷⁷Lu PRRT andcapecitabine (Xeloda; Genentech), an oral chemotherapy agent, with[¹⁷⁷Lu]DOTATATE alone has been started at Erasmus MC, Rotterdam in 2017(van Essen M, Krenning E P, Kam B L, de Herder W W, van Aken M O,Kwekkeboom D J. Report on short-term side effects of treatments with177Lu-octreotate in combination with capecitabine in seven patients withgastroenteropancreatic neuroendocrine tumours. Eur J Nucl Med MolImaging. 2008; 35:743-748).

Further studies on combination therapies, named peptide receptorchemoradionuclide therapy (PRCRT), have recently been published (Kong G,Callahan J, Hofman M S, et al. High clinical and morphologic responseusing 90Y-DOTA-octreotate sequenced with 177Lu-DOTA-octreotate inductionpeptide receptor chemoradionuclide therapy (PRCRT) for bulkyneuroendocrine tumours. Eur J Nucl Med Mol Imaging. 2017; 44:476-489).Similar “combined treatment approaches” will be carried out in the nearfuture to improve the efficiency of PSMA-targeted radioligand therapies.

In a further aspect, the present invention provides one or morecompounds or salts of the invention as disclosed herein above for use inmedicine.

Preferred uses in medicine are in nuclear medicine such as nucleardiagnostic imaging, also named nuclear molecular imaging, and/ortargeted radiotherapy of diseases associated with an overexpression,preferably of PSMA on the diseased tissue.

In a further aspect, the present invention provides a compound or saltof the invention as defined herein above for use in a method ofdiagnosing and/or staging cancer, preferably prostate cancer.

Preferred indications are the detection or staging of cancer, such as,but not limited high grade gliomas, lung cancer and especially prostatecancer and metastasized prostate cancer, the detection of metastaticdisease in patients with primary prostate cancer of intermediate-risk tohigh-risk, and the detection of metastatic sites, even at low serum PSAvalues in patients with biochemically recurrent prostate cancer. Anotherpreferred indication is the imaging and visualization of neoangiogensis.

In terms of medical indications to be subjected to therapy, especiallyradiotherapy, cancer is a preferred indication. Prostate cancer is aparticularly preferred indication.

In a further aspect, the present invention provides a compound or saltof the invention as defined herein above for use in a method ofdiagnosing and/or staging cancer, preferably prostate cancer.

As regards the embodiments characterized in this specification, inparticular in the claims, it is intended that each embodiment mentionedin a dependent claim is combined with each embodiment of each claim(independent or dependent) said dependent claim depends from. Forexample, in case of an independent claim 1 reciting 3 alternatives A, Band C, a dependent claim 2 reciting 3 alternatives D, E and F and aclaim 3 depending from claims 1 and 2 and reciting 3 alternatives G, Hand I, it is to be understood that the specification unambiguouslydiscloses embodiments corresponding to combinations A, D, G; A, D, H; A,D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B,D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C,D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C,F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependentclaims do not recite alternatives, it is understood that if dependentclaims refer back to a plurality of preceding claims, any combination ofsubject-matter covered thereby is considered to be explicitly disclosed.For example, in case of an independent claim 1, a dependent claim 2referring back to claim 1, and a dependent claim 3 referring back toboth claims 2 and 1, it follows that the combination of thesubject-matter of claims 3 and 1 is clearly and unambiguously disclosedas is the combination of the subject-matter of claims 3, 2 and 1. Incase a further dependent claim 4 is present which refers to any one ofclaims 1 to 3, it follows that the combination of the subject-matter ofclaims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well asof claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

In particular, the invention provides the subject matter summarized inthe following items.

-   1. A compound of formula (I), or a pharmaceutically acceptable salt    thereof,

-   -   wherein:    -   m is an integer of 2 to 6, preferably 2 to 4, more preferably 2;    -   n is an integer of 2 to 6, preferably 2 to 4, more preferably 2        or 4;    -   R^(1L) is CH₂, NH or O, preferably NH;    -   R^(2L) is C or P(OH), preferably C;    -   R^(3L) is CH₂, NH or O, preferably NH;    -   X¹ is selected from an amide bond, an ether bond, a thioether        bond, an ester bond, a thioester bond, a urea bridge, and an        amine bond, and is preferably an amide bond;    -   L¹ is a divalent linking group with a structure selected from an        oligoamide, an oligoether, an oligothioether, an oligoester, an        oligothioester, an oligourea, an oligo(ether-amide), an        oligo(thioether-amide), an oligo(ester-amide), an        oligo(thioester-amide), oligo(urea-amide), an        oligo(ether-thioether), an oligo(ether-ester), an        oligo(ether-thioester), an oligo(ether-urea), an        oligo(thioether-ester), an oligo(thioether-thioester), an        oligo(thioether-urea), an oligo(ester-thioester), an        oligo(ester-urea), and an oligo(thioester-urea), preferably with        a structure selected from an oligoamide and an        oligo(ester-amide),    -    which linking group may carry a group EDS;    -   X² is selected from an amide bond, an ether bond, a thioether        bond, an ester bond, a thioester bond, a urea bridge, and an        amine bond, and is preferably an amide bond;    -   R² is an optionally substituted aryl group or an optionally        substituted aralkyl group, which aryl group or aralkyl group may        be substituted on its aromatic ring with one or more        substituents selected from halogen, preferably I, and —OH;    -   R³ is an optionally substituted aryl group or an optionally        substituted aralkyl group, which aryl group or aralkyl group may        be substituted on its aromatic ring with one or more        substituents selected from halogen, preferably I, and —OH;    -   r is 0 or 1, preferably 1;    -   p is 0 or 1;    -   q is 0 or 1;    -    and preferably p+q=1;    -   R⁴ is selected from an optionally substituted aryl group and a        group EDS, which aryl group may be substituted on its aromatic        ring with one or more substituents selected from halogen,        preferably I, —OH and —NH₂;    -   X³ is selected from an amide bond, an ether bond, a thioether        bond, an ester bond, a thioester bond, a urea bridge, an amine        bond, and a group of the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) andthe other marked bond attaches X³ to the remainder of the compound offormula (I);

-   -    and is preferably an amide bond;    -   R^(M) is a marker group which comprises a chelating group        optionally containing a chelated non-radioactive or radioactive        cation;

-    and wherein the group EDS is contained at least once in the    compound of formula (I) and has a structure selected from (E-1A),    (E-1B), (E-2A) and (E-2B):

-    wherein-   marks the bond which attaches the group EDS, to the remainder of the    compound of formula (I);-    s is 1, 2 or 3, preferably 1 or 2, and more preferably 1;-    t is 1, 2 or 3, preferably 1 or 2, and more preferably 2;-    R^(5A) is, independently for each occurrence for s>1, an electron    withdrawing substituent, which is preferably selected from —NO₂ and    —COOH, and which is more preferably —COOH, and wherein the bond    between R^(5A) and the phenyl ring indicates that the s groups    R^(5A) replace s hydrogen atoms at any position on the phenyl ring;-    R^(5B) is, independently for each occurrence for s>1, a substituent    carrying an electron lone pair at the atom directly attached to the    phenyl ring shown in formula (E-1B), which substituent is preferably    selected from —OH and —NH₂, and which is more preferably —NH₂, and    wherein the bond between R^(5B) and the phenyl ring indicates that    the s groups R^(5B) replace s hydrogen atoms at any position on the    phenyl ring;-    R^(6A) is, independently for each occurrence for t>1, an electron    withdrawing substituent, which is preferably selected from —NO₂ and    —COOH, and which is more preferably —COOH, and wherein the bond    between R^(6A) and the phenyl ring indicates that the t groups    R^(6A) replace t hydrogen atoms at any position on the phenyl ring;    and-    R^(6B) is, independently for each occurrence for t>1, a substituent    carrying an electron lone pair at the atom directly attached to the    phenyl ring shown in formula (E-1B), which substituent is preferably    selected from —OH and —NH₂, and which is more preferably —OH, and    wherein the bond between R^(6B) and the phenyl ring indicates that    the t groups R^(6B) replace t hydrogen atoms at any position on the    phenyl ring.-   2. The compound or salt of item 1, wherein m is 2, n is 2 or 4,    R^(1L) is NH, R^(2L) is C, and R^(3L) is NH.-   3. The compound or salt of item 1 or 2, wherein n is 2.-   4. The compound or salt of any of items 1 to 3, wherein X¹ is an    amide bond.-   5. The compound or salt of item 4, wherein n is 2 and X¹ is an amide    bond with the carbon atom of the amide bond —C(O)—NH— being attached    to the group —(CH₂)_(n)—.-   6. The compound or salt of any of items 1 to 5, wherein L¹ is a    divalent linking group with a structure selected from an oligoamide    which comprises a total of 1 to 5, more preferably a total of 1 to    3, and most preferably a total of 1 or 2 amide bonds within its    backbone, and an oligo(ester-amide) which comprises a total of 2 to    5, more preferably a total of 2 to 3, and most preferably a total of    2 amide and ester bonds within its backbone, which linking group may    carry a group EDS.-   7. The compound or salt of item 6, wherein L¹ represents a divalent    linking group with an oligoamide structure which comprises 1 or 2    amide bonds within its backbone, which linking group may carry a    group EDS.-   8. The compound or salt of any of items 1 to 7, wherein the linking    group L¹ carries one group EDS.-   9. The compound or salt of any of items 1 to 8, wherein X² is an    amide bond.-   10. The compound or salt of item 9, wherein X² is an amide bond with    the nitrogen atom of the amide bond —C(O)—NH— being attached to L¹.-   11. The compound or salt of any of items 1 to 10, wherein the moiety    —X²-L¹-X¹— in formula (I) has a structure selected from:

*—C(O)—NH—R⁷—NH—C(O)—R⁸—C(O)—NH—  (L-1),

*—C(O)—NH—R^(9A)—NH—C(O)—R^(10A)—C(O)—NH—R^(10A)—NH—C(O)—  (L-2A), and

*—C(O)—NH—R^(9B)—C(O)—NH—R^(10B)—C(O)—NH—R^(11B)—NH—C(O)—  (L-2B);

-    wherein the amide bond marked with * is attached to the carbon atom    carrying R² in formula (I), and wherein-    R⁷, R⁸, R^(9A), R^(9B), R^(11A) and R^(11B) are independently    selected from optionally substituted C2 to C10 alkanediyl,    preferably optionally substituted linear C2 to C10 alkanediyl, which    alkanediyl groups may each be substituted by one or more    substitutents independently selected from —OH, —OCH₃, —COOH,    —COOCH₃, —NH₂, —NHC(NH)NH₂, and a group EDS, and-    R^(10A) and R^(10B) are selected from optionally substituted C2 to    C10 alkanediyl, preferably optionally substituted linear C2 to C10    alkanediyl, and optionally substituted C6 to C10 arenediyl,    preferably phenylene, which alkanediyl and arenediyl group may each    be substituted by one or more substitutents independently selected    from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, —NHC(NH)NH₂, and a group EDS.    R^(10A) is preferably optionally substituted C2 to C10 alkanediyl,    more preferably optionally substituted linear C2 to C10 alkanediyl    as defined above. R^(10B) is preferably optionally substituted C6 to    C10 arenediyl as defined above, more preferably a phenylene group,    e.g. para-phenylene group.-   12. The compound or salt of item 11, wherein the total number of    carbon atoms in R⁷ and R⁸ of formula (L-1) is 6 to 20, more    preferably 6 to 16, without carbon atoms contained in optional    substituents, that the total number of carbon atoms in R^(9A),    R^(10A) and R^(11A) of formula (L-2A) is 6 to 20, more preferably 6    to 16, without carbon atoms contained in optional substituents, and    that the total number of carbon atoms in R^(9B), R^(10B) and R^(11B)    of formula (L-2B) is 6 to 20, more preferably 6 to 16, without    carbon atoms contained in optional substituents.-   13. The compound or salt of item 11 or 12, wherein-    the moiety —X²-L¹-X¹— has a structure (L-1) and R⁸ carries a group    EDS as at least one substituent, or-    the moiety —X²-L¹-X¹— has a structure (L-2A) and R^(10A) carries a    group EDS as at least one substituent.-   14. The compound or salt of item 7, wherein the moiety —X²-L¹-X¹—    has a structure selected from:

*—C(O)—NH—CH(COOH)—R¹²—NH—C(O)—R¹³—C(O)—NH—  (L-3),

*—C(O)—NH—CH(COOH)—R¹⁴—NH—C(O)—R¹⁵—C(O)—NH—R¹⁶—CH(COOH)—NH—C(O)—  (L-4), and

*—C(O)—NH—CH(COOH)—R¹⁷—C(O)—NH—R¹⁸—C(O)—NH—R¹⁹—CH(COOH)—NH—C(O)—  (L-5);

-    wherein the bond marked with * is attached to the carbon atom    carrying R² in formula (I),-    R¹² and R¹⁴ are independently selected from linear C2 to C6    alkanediyl, preferably from linear C3 to C6 alkanediyl,-    R¹³ is a linear C2 to C10 alkanediyl, preferably a linear C4 to C8    alkanediyl,-    R¹⁵ and R¹⁶ are independently selected from linear C2 to C6    alkanediyl, preferably from linear C2 to C4 alkanediyl,-    and wherein each of R¹³ and R¹⁵ may carry one group EDS as a    substituent, and more preferably each of R¹³ and R¹⁵ carries one    group EDS as a substituent,-    R¹⁷ is a linear C2 to C6 alkanediyl, preferably a linear C2 to C4    alkanediyl,-    R¹⁸ is a phenylene group, e.g. a para-phenylene group, and-    R¹⁹ is a linear C2 to C6 alkanediyl, preferably a linear C2 to C4    alkanediyl.-   15. The compound or salt of item 14, wherein the total number of    carbon atoms in R¹² and R¹³ in formula (L-3), without carbon atoms    contained in the group EDS as substituent, is 6 to 16, more    preferably 6 to 14, and the total number of carbon atoms in R¹⁴, R¹⁵    and R¹⁶ in formula (L-4), without carbon atoms contained in the    group EDS as substituent, is 6 to 16, more preferably 6 to 14.-   16. The compound or salt of any of items 1 to 15, wherein R² is an    optionally substituted aralkyl group selected from optionally    substituted —CH₂-phenyl and optionally substituted —CH₂-naphtyl,    more preferably optionally substituted —CH₂-(2-naphtyl), wherein the    phenyl and the naphtyl group are optionally substituted with a    substituent selected from halogen, preferably I, and —OH.-   17. The compound or salt of item 16, wherein R² is an aralkyl group    of the formula —CH₂-naphthyl, more preferably —CH₂-(2-naphthyl).-   18. The compound or salt of any of items 1 to 17, wherein R³ is an    optionally substituted aralkyl group selected from optionally    substituted —CH₂-phenyl and optionally substituted —CH₂-naphtyl,    more preferably optionally substituted —CH₂-phenyl, wherein the    phenyl and the naphtyl group are optionally substituted with a    substituent selected from halogen, preferably I, and —OH.-   19. The compound or salt of item 18, wherein R³ is an aralkyl group    of the formula —CH₂-phenyl, wherein the phenyl ring is substituted    with with one substituent which is —OH, or with a combination of one    substituent —OH and one substituent —I.-   20. The compound or salt of any of items 1 to 15,-    wherein R² is a group of the formula

-    and R³ is a group of the formula

-    wherein    marks the bond which attaches R² and R³, respectively, to the    remainder of the compound of formula (I).-   21. The compound or salt of item 20,-    wherein R² is a group of the formula

-    and R³ is a group of the formula

-    wherein    marks the bond which attaches R² and R³, respectively, to the    remainder of the molecule.-   22. The compound or salt of any of items 1 to 21, wherein r is 1.-   23. The compound or salt of any of items 1 to 22, wherein p is 0 and    q is 1.-   24. The compound or salt of any of items 1 to 23, wherein R⁴ is    selected from phenyl, optionally naphtyl, and a group EDS.-   25. The compound or salt of any of item 24, wherein R⁴ is selected    from naphtyl, more preferably 2-naphthyl and a group EDS.-   26. The compound or salt of any of items 1 to 25, wherein X³ is an    amide bond or a group of the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) andthe other marked bond attaches X³ to the remainder of the molecule.

-   27. The compound or salt of item 26, wherein X³ is an amide bond    —C(O)—NH— with the carbon atom being attached to R^(M).-   28. The compound or salt of any of items 1 to 27, wherein R^(M) is a    chelating group optionally containing a chelated non-radioactive or    radioactive cation.-   29. The compound or salt of any of items 1 to 28, wherein the    chelating group comprises at least one of    -   (i) a macrocyclic ring structure with 8 to 20 ring atoms of        which 2 or more, preferably 3 or more, are selected from oxygen        atoms, sulfur atoms and nitrogen atoms; and    -   (ii) an acyclic, open chain chelating structure with 8 to 20        main chain atoms of which 2 or more, preferably 3 or more are        heteroatoms selected from oxygen atoms, sulfur atoms and        nitrogen atoms.-   30. The compound or salt of any of items 1 to 29, wherein the    chelating group is a residue of a chelating agent selected from    bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane    (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA),    4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA),    N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide    (DFO),    4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicycle[6.6.2]hexadecan    (DO2A) 1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid    (DOTA), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-triacetic    acid]-pentanedioic acid (DOTAGA),    N,N′-dipyridoxylethylendiamine-N,N′-diacetate-5,5′-bis(phosphat)    (DPDP), diethylenetriaminepentaacetic acid (DTPA),    ethylenediamine-N,N′-tetraacetic acid (EDTA),    ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid    (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid    (HBED), hydroxyethyldiaminetriacetic acid (HEDTA),    1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate    (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC),    1,4,7-triazacyclononan-1-succinic acid-4,7-diacetic acid (NODASA),    1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane    (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA),    4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane    (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid    (TETA), terpyridin-bis(methyleneamintetraacetic acid (TMT),    1,4,7,10-tetraazacyclotridecan-N,N′,N″,N′″-tetraacetic acid (TRITA),    and triethylenetetraaminehexaacetic acid (TTHA),    N,N′-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6    (H₂macropa) and    4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl}heptanedioic    acid    bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-amide]    (THP);-    which residue is provided by covalently binding a carboxyl group    contained in the chelating agent to the remainder of the compound    via an ester or an amide bond, more preferably an amide bond.-   31. The compound or salt of item 30, wherein the chelating agent is    selected from DOTA and DOTAGA.-   32. The compound or salt of item 30 or 31, wherein X³ is the amide    bond attaching the chelating group to the remainder of the molecule.-   33. The compound or salt of item 32, wherein R^(M)—X³— is a group of    the formula

-    wherein the bond marked with    is attached to the remainder of the compound of formula (I), and    wherein the chelating group may contain a chelated non-radioactive    or radioactive cation.-   34. The compound or salt of any of items 1 to 33, wherein the    chelating group comprises a chelated cation, preferably a chelated    radioactive cation selected from the cations of ⁴⁴Sc, ⁴⁷Sc, ⁵¹Cr,    ^(52m)Mn, ⁵⁸Co, ⁵²Fe, ⁵⁶Ni, ⁵⁷Ni, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁸Ga,    ⁶⁷Ga, ⁸⁹Zr, ⁹⁰Y, ⁸⁹Y, ^(94m)Tc, ^(99m)Tc, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag,    ^(110m)In, ¹¹¹In, ^(113m)In, ^(114m)In, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te,    ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶¹Tb, ¹⁶⁶Ho,    ¹⁶⁵Dy, ¹⁶⁹Er, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷²Tm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹¹Pt,    ¹⁹⁷Hg, ¹⁹⁸Au, ¹⁹⁹Au, ²¹²Pb, ²⁰³Pb, ²¹¹At, ²¹²Bi ²¹³Bi, ²²³Ra, ²²⁵Ac,    and ²²⁷Th, or a cationic molecule comprising ¹⁸F, such as    ¹⁸F-[AIF]²⁺.-   35. The compound or salt of item 34, wherein the chelating group    comprises a chelated cation selected from the cations of ⁴⁴Sc, ⁴⁷Sc,    ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁹⁰Y, ¹¹¹In, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁸Re, ²¹²Pb,    ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th or a cationic molecule comprising    ¹⁸F.-   36. The compound or salt of any of items 1 to 35, wherein the    compound of formula (I) contains one or two groups EDS.-   37. The compound or salt of item 36, wherein the compound of    formula (I) either contains one group EDS which is carried by the    linking group L¹, or contains two groups EDS, one being represented    by R⁴ and one being carried by L¹.-   38. The compound or salt of any of items 1 to 37, wherein, in the    group EDS (E-1A), the substituents R^(5A) are the same for s>1 and    are selected from —NO₂ and —COOH; and wherein, in the group EDS    (E-2A), the substituents R^(6A) are the same for t>1 and are    selected from —NO₂ and —COOH.-   39. The compound or salt of any of items 1 to 38, wherein, in the    group EDS (E-1B), the substituents R^(5B) are the same for s>1 and    are selected from —OH and —NH₂; in the group EDS (E-2B), the    substituents R^(6B) are the same for t>1 and are selected from —OH    and —NH₂.-   40. The compound or salt of any of items 1 to 39, which contains a    group EDS which has the formula (E-2A):

-    wherein    marks the bond which attaches the group EDS to the remainder of the    compound of formula (I); and-    t is 1 or 2, and R^(6A) is selected from —NO₂ and —COOH.-   41. The compound of any of items 1 to 38, wherein the group EDS has    the formula (E-3)

-    wherein    marks the bond which attaches the group EDS to the remainder of the    compound of formula (I).-   42. The compound of any of items 1 to 41, which has the following    formula (Ia), or a pharmaceutically acceptable salt thereof:

-    wherein n, X¹, L¹, X², R², R³, R⁴, q, p, X³ and R^(M) are defined    as in the preceding items, and wherein the group EDS is contained at    least once and has a structure as defined in the preceding items.-   43. The compound of item 42, which has the following formula (Ib),    or a pharmaceutically acceptable salt thereof:

-    wherein n, X¹, L¹, X², R², R³, R⁴, X³ and R^(M) are defined as in    the preceding items, and wherein the group EDS is contained at least    once and has a structure as defined in the preceding items.-   44. The compound of any of item 43, which has the following formula    (Ic), or a pharmaceutically acceptable salt thereof:

-    wherein n, X¹, L¹, X², R⁴, X³ and R^(M) are defined as in the    preceding items, and wherein the group EDS is contained at least    once and has a structure as defined in the preceding items.-   45. The compound of item 44, which has the following formula (Id) or    (Ie), or a pharmaceutically acceptable salt thereof:

-    wherein R^(9A), R^(10A), R^(11A), R⁴, X³ and R^(M) are defined as    in the preceding items, and wherein either (i) R4 is a group EDS    with a structure as defined in the preceding items, or (ii) R^(10A)    carries one group EDS with a structure as defined in the preceding    items, or both (i) and (ii) apply;

-    wherein R^(9B), R^(10B), R^(11B), R⁴, X³ and R^(M) are defined in    the preceding items, and wherein R⁴ is a group EDS with a structure    as defined in the preceding items.-   46. The compound of item 45, which has the following formula (If) or    (Ig), or a pharmaceutically acceptable salt thereof:

-    wherein R^(9A), R^(10A), R^(11A), R⁴, X³ and R^(M) are defined as    in the preceding items, and wherein either (i) R4 is a group EDS    with a structure as defined in the preceding items, or (ii) R^(10A)    carries one group EDS with a structure as defined in the preceding    items, or both (i) and (ii) apply;

-    wherein R^(9B), R^(10B), R^(11B), R⁴, X³ and R^(M) are defined as    in the preceding items, and wherein R⁴ is a group EDS with a    structure as defined in the preceding items.-   47. The compound or salt thereof of any item 1, wherein said    compound or salt has one of the following formulae:

-   48. A pharmaceutical or diagnostic composition comprising or    consisting of one or more compounds or salts in accordance with any    one of items 1 to 47.-   49. A compound or salt in accordance with any one of items 1 to 47    for use in a method of diagnosing and/or treating    -   (a) cancer including prostate cancer; or    -   (b) neoangiogenesis/angiogenesis.

The figures show:

FIG. 1 : Web chart of characteristics for [^(nat/177)Lu]PSMA I&T and[^(nat/177)Lu]PSMA-62 and [^(nat/177)Lu]PSMA-66.

FIG. 2 : Externalization kinetics of selected ¹⁷⁷Lu-labeled PSMAinhibitors from LNCaP cells. 1.25*10⁵ cells/well were incubated 1 h withthe respective radioligand (c=1.0 nm) at 37° C. in DMEM-solution (5%BSA). Then, the supernatant was removed and once washed withDMEM-solution (5% BSA, 37° C.). Afterwards, either A) only DMEM-solution(5% BSA) or B) blockade DMEM-solution (5% BSA, 10 μm 2-PMPA) were addedfor replacement. The total cellular internalized activity at t=0 min wascorrected for non-specific binding (10 μm 2-PMPA) and normalized to100%. All data are expressed as mean±SD (n=3).

FIG. 3 : Biodistribution (in % ID/g) of 2.5 to 3.0 MBq (0.15 to 0.25nmol) of [¹⁷⁷Lu]PSMA-66 and [¹⁷⁷Lu]PSMA I&T in LNCaP-tumor bearing CB-17SCID mice (n=4, respectively).

FIG. 4 : Maximum intensity projection (MIP) of a pPET scan inLNCaP-tumor bearing CB-17 SCID mice after injection of approx. 10.3 MBq(0.19 nmol tracer) of [⁶⁸Ga]PSMA-36 (dynamic scan, summed up frames 1 to1.5 h p.i.) (top left). TACs (logarithmic plot) in % ID/mL of[⁶⁸Ga]PSMA-36 derived from dynamic PET data (90 min acquisition time,OSEM 3D reconstruction) in a LNCaP-tumor bearing CB-17 SCID mouse ofblood pool (heart), kidney, tumor, muscle, lacrimal- and salivary gland.

FIG. 5 : Maximum intensity projection (MIP) of μPET scans in LNCaP-tumorbearing CB-17 SCID mice after injection of approx. 11 and 13 MBq (0.15to 0.25 nmol tracer) of the ⁶⁸Ga-labeled PSMA inhibitor PSMA-62 andPSMA-66, respectively (dynamic scan, summed up frames 1 to 1.5 h p.i.)(top left). TACs (logarithmic plot) in % ID/mL of the respective⁶⁸Ga-labeled PSMA inhibitor derived from dynamic PET data (90 minacquisition time, OSEM 3D reconstruction) in LNCaP-tumor bearing CB-17SCID mice of blood pool (heart), kidney, tumor and muscle for both⁶⁸Ga-labeled tracer.

FIG. 6 : Biodistribution (in % ID/g) of 2.5 to 6.0 MBq (0.15 to 0.25nmol) of [¹⁷⁷Lu]PSMA-62, [¹⁷⁷Lu]PSMA-66, [¹⁷⁷Lu]PSMA-71 and [¹⁷⁷Lu]PSMAI&T in LNCaP-tumor bearing CB-17 SCID mice (n=4, respectively).

The examples illustrate the invention.

EXAMPLE 1: MATERIALS AND METHODS 1. General Information

The Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected aminoacid analogs were purchased from Bachem (Bubendorf, Switzerland) or IrisBiotech (Marktredwitz, Germany). The 2-chlorotrityl chloride (2-CTC)resin was obtained from PepChem (Tübingen, Germany). Chematech (Dijon,France) delivered the chelator DOTAGA-anhydride. PSMA-DKFZ-617 waspurchased from ABX advanced chemical compounds (Radeberg, Germany). Allnecessary solvents and other organic reagents were purchased from eitherAlfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany) or VWR(Darmstadt, Germany). Solid phase synthesis of the peptides was carriedout by manual operation using an Intelli-Mixer syringe shaker (Neolab,Heidelberg, Germany). Analytical reversed-phase high performance liquidchromatography (RP-HPLC) was performed on a Nucleosil 100 C18 column (5μm, 125×4.0 mm, CS GmbH, Langerwehe, Germany) using a Shimadzu gradientRP-HPLC System (Shimadzu Deutschland GmbH, Neufahrn, Germany). Analysisof the peptides was performed by applying different gradients of 0.1%(v/v) trifluoroacetic acid (TFA) in H₂O (solvent A) and 0.1% TFAtogether with (v/v) in acetonitrile (MeCN) (solvent B) with a constantflow of 1 mL/min (specific gradients are cited in the text). TheShimadzu SPD 20 A prominence UV/VIS detector (Shimadzu Deutschland GmbH)was used at A=220 nm and 254 nm. HSA binding was determined using aChiralpak HSA (5 μm, 50×3 mm) analytical column connected to a ChiralpakHSA (5 μm, 10×3 mm) guard cartridge (Daicel Chemical Industries)purchased from Chiral Technologies Europe (Illkirch, France). Non-linearregression for the HSA binding was performed using OriginPro 2016G(Northampron, USA). Retention times t_(R) as well as the capacityfactors K′ are cited in the text. Preparative RP-HPLC of the peptideswas achieved on a Shimadzu RP-HPLC system using a Multospher 100 RP 18-5column (250×20 mm, CS GmbH) with a constant flow of 5 mL/min. Analyticaland preparative Radio RP-HPLC of the radioiodinated reference ligand wasperformed using a Nucleosil 100 C18 column (5 μm, 125×4.0 mm).Radioactivity was detected through connection of the outlet of theUV-photometer to a NaI(Tl) well-type scintillation counter from EG&GOrtec (Munich, Germany). The ⁶⁸Ga- and ¹⁷⁷Lu-labeled compounds wereanalyzed as published previously [1, 2]. Electrospray ionization massspectrometry (ESI-MS) spectra were acquired on an expression^(L) CMSmass spectrometer (Advion Ltd., Harlow, UK) and on a Varian 500-MS ITmass spectrometer (Agilent Technologies, Santa Clara, USA). For theBradford-Assay a V-630 UV-Vis spectrophotometer from JASCO Germany GmbH(Gross-Umstadt, Germany) was used and centrifugation of the S9-fractionswas performed in an Avanti JXN-26 centrifuge from Beckman Coulter GmbH(Krefeld, Germany). The centrifugation of the radioactive S9-metaboliteassays was performed using a Heraeus PICO 17 centrifuge from ThermoFisher Scientific Messtechnik GmbH (Munich, Germany). NMR Data wereobtained applying 300 K using an AV 300 (300 MHz) or an AV 400 (400 MHz)from Bruker (Billerica, USA). The incubation of the S9-fractions for exvivo metabolite analysis was performed in a Biometra UNO Thermoblock(Biometra, Göttingen, Deutschland).

2. Synthesis Protocols (SP)

SP-1: 2-CTC-resin loading: 2-CTC-resin (1.6 mmol/g) is loaded withFmoc-AA-OH (1.5 eq.) in anhydrous dichloromethane (DCM) withN,N-Diisopropylethylamine (DIPEA) (4.5 eq.) at room temperature (RT) for2 h. The remaining tritylchloride is capped by addition of 2 mL/gmethanol (MeOH) for 15 min. After that, the resin is filtered andthoroughly washed with DCM (2^(x)), with dimethylformamide (DMF) (2^(x))and MeOH (2^(x)), respectively and stored under vacuum overnight. Theloading is determined using the weight differences:

$\frac{\left. {m_{total} - m_{{net}{weight}}} \right) \times 1000}{\left( {M_{As} - M_{HCl}} \right) \times m_{{weight}{of}{resin}}} = {{mmol}/g}$Formula1.Determinationofresin − loading : m_(total) : massofloadedresin(Fmoc − AA − OHandHCI); M_(As) : molarmassofaminoacid; m_(net)weight : massofusedresin; M_(HCl) : molarmassofhydrochloricacid

SP-2: Peptide synthesis via TBTU/HOBt coupling: A solution of Fmoc-AA-OH(2.0 eq.), N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uroniumtetrafluoroborate (TBTU) (2.0 eq.), N-Hydroxybenzotriazole (HOBt) (2.0eq.), DIPEA (4.5 eq.) in DMF (8 ml/g resin) was added to the resin-boundfree amine peptide and shaken for 2 h at RT and washed with DMF (6^(x)).The coupling with secondary or aromatic amines was performed employing adifferent protocol. Fmoc-AA-OH (3.0 eq.) was dissolved in DMF (8 mL/gresin) together with1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluoro-phosphate (HATU) (3.0 eq.),1-Hydroxy-7-azabenzotriazol (HOAt) (3.0 eq.) and DIPEA (6.0 eq.) andstirred for 15 min. The pre-activated solution was added to the resinbound peptide and shaken for 2 h at RT. After completion of thereaction, the resin was washed with DMF (6^(x)). In general, allpeptidic scaffolds were synthesized as previously described (Weineisen,M.; Schottelius, M.; Simecek, J.; Eiber, M.; Schwaiger, M.; Wester, H.Development and first in human evaluation of PSMA I&T—A ligand fordiagnostic imaging and endoradiotherapy of prostate cancer. Journal ofNuclear Medicine 2014, 55, 1083-1083; Weineisen, M.; Simecek, J.;Schottelius, M.; Schwaiger, M.; Wester, H.-J. Synthesis and preclinicalevaluation of DOTAGA-conjugated PSMA ligands for functional imaging andendoradiotherapy of prostate cancer. EJNMMI research 2014, 4, 1).

SP-3: On-resin Fmoc-deprotection: The resin-bound Fmoc-protected peptidewas treated with 20% piperidine in DMF (v/v) for 5 min and a second timefor 15 min. Afterwards, the resin was washed thoroughly with DMF(8^(x)).

SP-4: On-resin Dde-deprotection: TheN-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl) (Dde) protectedpeptide (1.0 eq.) was dissolved in a solution of 2.0% hydrazinemonohydrate (N₂H₄.H₂O) in DMF (v/v). After 15 min, the deprotectedpeptide, if bound to resin, was washed with DMF (6^(x)) or precipitatedin diethyl ether (Et₂O) to give the crude product. If Fmoc- andDde-protecting groups were present and only Dde-deprotection wasnecessary, the resin-loaded peptide was treated with a solutioncontaining NH₂OH.HCl (630 mg), imidazole (460 mg), DCM (0.5 mL), DMF(0.5 mL) and N-methyl-2-pyrrolidone (NMP) (2.5 mL) for 3 h at RT.Afterwards, the resin-loaded peptide was washed with DMF (6^(x)).

SP-5: On-resin Alloc/Allyl-deprotection: The Alloc/Allyl-protectinggroup was removed from the resin-bound peptide using a solution of DCM(6.0 mL) containing triisopropylsilane (TIPS) (50.0 eq.) and(triphenyl)palladium(0) (Pd(PPh₃)₄) (0.3 eq.). The resin was treatedwith this solution for 1.5 h at RT. Finally, the resin was washed withDCM (3^(x)) to remove the Pd(PPh₃)₄.

SP-6: tBu/Boc deprotection: Removal of the tert-butyl(tBu)/tert-butyloxycarbonyl (Boc)-protecting groups was carried out bydissolving the crude product in TFA (approx. 500 μL) and stirring for 40min at RT. Afterwards, the TFA was almost completely removed usingnitrogen stream. After precipitation in Et₂O, the crude product wascentrifuged and the supernatant removed. The dried pellet was furtherused for the following synthesis-steps.

SP-7.1: A) Peptide cleavage from the resin with preservation ofside-chain protecting groups: The fully protected, resin-bound peptidewas dissolved in a mixture of DCM/trifluoroethanol (TFE)/acetic acid(AcOH) (6/3/1; v/v/v) and shaken for 30 min. The solution was filteredoff and the resin was dissolved in another cleavage solution for another30 min. The fractions were combined and the solvent was concentratedunder reduced pressure. The filtrate was redissolved in toluene andconcentrated under reduced pressure to remove the AcOH. Precipitation inwater or Et₂O resulted in the crude, side chain protected peptide.

SP-7.2: B) Peptide cleavage from the resin with concurrent deprotectionof all acid labile protecting groups: The fully protected, resin-boundpeptide was dissolved in a mixture of TFA/TIPS/water (95/2.5/2.5; v/v/v)and shaken for 30 min. The solution was filtered off and the resin wastreated in the same way for another 30 min. Afterwards, the fractionswere combined and the solvent was concentrated under a constant flow ofnitrogen. The crude peptide was precipitated in Et₂O and left to dryovernight.

SP-8: Deacetylation of carbohydrate-moieties: Deacetylation wasaccomplished by dissolving the PSMA inhibitor in MeOH containing KCN(0.5 eq.) (Herzig, J.; Nudelman, A.; Gottlieb, H. E.; Fischer, B.Studies in sugar chemistry. 2. A simple method for O-deacylation ofpolyacylated sugars. The Journal of Organic Chemistry 1986, 51, 727-730)with concomitant stirring overnight at RT. The final product waspurified by RP-HPLC.

SP-9: Preparation of non-radioactive metal-complexed PSMA inhibitors:

SP-9.1: ^(nat)Ga-compounds: For the preparation of the^(nat)Ga^(III)-complexes, a 2.0 mm aqueous (aq.) solution of the PSMAinhibitor (50 μL) and a 2.0 mm aq. solution of Ga(NO₃)₃ (50 μL) weremixed and heated at 40° C. for 30 min. The chelate formation wasassessed using RP-HPLC and ESI-MS. The resulting 1.0 mm solution wasdiluted and used for in vitro IC₅₀ determination and HSA binding.

SP-9.2: ^(nat)Lu-compounds: The corresponding ^(nat)Lu^(III)-complexeswere prepared from a 2.0 mm aqueous solution of the PSMA inhibitor witha 2.5 molar excess of LuCl₃ (20 mm aq. solution) and heated to 95° C.for 30 min. After cooling, the ^(nat)Lu^(III)-chelate formation wasconfirmed using RP-HPLC and ESI-MS. The resulting 1.0 mm aqueoussolutions of the respective ^(nat)Lu-complexes were then diluted andused in the in vitro IC₅₀ studies without further processing.

3. Building Blocks for PSMA-36 and the EuE Based PSMA InhibitorsDi-tert-butyl(((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate

((OtBu)KuE(OtBu)₂) (1): The synthesis of the tert-butyl-protectedLys-urea-Glu binding motif (EuK) was synthesized as previously describedby solution phase synthesis [3]. In short, a solution of DCM containingL-di-tert-butyl-glutamate-HCl (2.0 g, 7.71 mmol, 1.0 eq.) was cooled onice for 30 min and afterwards treated with trimethylamine (TEA) (2.69mL, 19.28 mmol, 2.5 eq.) and 4-(dimethylamino)pyridine (DMAP) (3.3 mg,0.3 mmol, 0.04 eq.). After additional stirring for 5.0 min,1,1′-carbonyldiimidazole (CDI) (1.38 g, 8.84 mmol, 1.1 eq.) wasdissolved in DCM and slowly added over a period of 30 min. The reactionmixture was further stirred overnight and enabled to warm to RT. Thereaction was stopped using saturated (sat.) NaHCO₃ solution (8 mL) withconcomitant washing steps of water (2^(x)) and brine (2^(x)) and driedover sat. Na₂SO₄ solution. The remaining solvent was removed in vacuoand the crude product (S)-Di-tert-butyl2-(1H-imidazole-1-carboxamido)pentanedioate used without furtherpurification. RP-HPLC (10 to 90% B in 15 min): t_(R)=12.2 min; K′=5.8.Calculated monoisotopic mass (C₁₇H₂₇N₃O₅): 353.4; found: m/z=376.1[M+Na]⁺. The crude product (S)-Di-tert-butyl2-(1H-imidazole-1-carboxamido)pentanedioate (2.72 g, 7.71 mmol, 1.0 eq.)was dissolved in 1,2-dichloroethane (DCE) and cooled on ice for 30 min.To this solution was added TEA (2.15 mL, 15.42 mmol, 2.0 eq.) andH-Lys(Cbz)-OtBu.HCl (2.87 g, 7.71 mmol, 1.0 eq.) and the solutionstirred overnight at 40° C. The remaining solvent was evaporated and thecrude product purified using silica gel flash-chromatography with aneluent mixture containing ethyl acetate (EtOAc)/hexane/TEA (500/500/0.8;v/v/v). After removal of the solvent,(9R,13S)-tri-tert-butyl-3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazapentadecane-9,13,15-tricarboxylate was obtained as colorlessoil. RP-HPLC (40 to 100% B in 15 min): t_(R)=14.5 min; K′=6.25.Calculated monoisotopic mass (C₃₂H₅₁N₃O₉)=621.8; found: m/z=622.3[M+H]⁺. To synthesize (OtBu)KuE(OtBu)₂ (1),(9R,13S)-tri-tert-butyl-3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazapentadecane-9,13,15-tricarboxylate (3.4 g, 5.47 mmol, 1.0 eq.)was dissolved in ethanol (EtOH) (75 mL) and palladium on activatedcharcoal (0.34 g, 0.57 mmol, 0.1 eq.) (10%) was given to this solution.The reaction mixture containing flask was initially purged with hydrogenstream and the solution allowed to stir overnight at RT under lighthydrogen-pressure (balloon). The crude product was purified throughcelite and the solvent evaporated in vacuo. The desired product 1 wasobtained as a waxy solid (1.9 g, 3.89 mmol, 71.6% yield). RP-HPLC (10 to90% B in 15 min): t_(R)=12.6 min; K′=6.4. Calculated monoisotopic mass(C₂₄H₄₅N₃O₇)=487.6; found: m/z=488.3 [M+H]⁺, 510.3 [M+Na]⁺.

(S)-5-(tert-butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-oxopentanoicacid ((OtBu)EuE(OtBu)₂) (2)

The synthesis of the tert-butyl-protected Glu-urea-Glu binding motif(EuE) was similarly synthesized as described for 1 [3] usingH-L-Glu(OBzl)-OtBu.HCl instead of H-L-Lys(Cbz)-OtBu.HCl. The desiredproduct was obtained as waxy and strongly hygroscopic solid (4.10 g,8.39 mmol, 84% yield). RP-HPLC (10 to 90% B in 15 min): t_(R)=11.3 min;K′=7.69. Calculated monoisotopic mass (C₂₃H₄₉N₂O₉)=488.3; found:m/z=489.4 [M+H]⁺, 516.4 [M+Na]⁺.

(S)-NHFmoc-Asu(OtBu)-OBzl (5): To a solution of (S)-Fmoc-Asu(OtBu)-OH(50 mg, 107.0 μmol, 1.0 eq.) in DMF was added HOAt (21.8 mg, 0.16 mmol,1.5 eq.), HATU (61.0 mg, 161.0 μmol, 1.5 eq.) and DIPEA (73.2 μL, 0.48mmol, 4.5 eq.). After 15 min of stirring at RT, benzyl alcohol (22.2 μL,0.32 mmol, 3.0 eq.) was further added and the solution stirredovernight. Finally, the solvent was removed in vacuo. Completion ofreaction of 5 was analyzed by RP-HPLC (10 to 90% B in 15 min):t_(R)=17.1 min K′=7.55. Calculated monoisotopic mass for 5(C₃₄H₃₉NO₆)=557.28; found: m/z=580.7 [M+Na]⁺.

(S)-NHFmoc-Asu-OBzl (6): tBu deprotection of the crude product 5 wasperformed with a stirring mixture (v/v) of TFA (95%) and DCM (5%) at RTfor 45 min. After evaporation of the solvent, the crude product 6 waspurified using preparative RP-HPLC (60 to 80% B in 15 min): t_(R)=9.3min; K′=8.9. Calculated monoisotopic mass for 6 (C₃₀H₃₁NO₆)=501.22;found m/z=524.5 [M+Na]⁺.

OBzl-(S)-Fmoc-Asu[(OtBu)KuE(OtBu)₂] (7): To a solution of 6 (51.8 mg,10.3 μmol, 1.0 eq.) in DMF was added HOBt (20.9 mg, 0.15 mmol, 1.5 eq.),TBTU (36.3 mg, 15.5 μmol, 1.5 eq.) and DIPEA (79.4 μL, 59.7 mg, 0.46mmol, 4.5 eq.). After 15 min stirring, 1 (75.6 mg, 15.5 μmol, 1.5 eq.)was added and further stirred for 20 h at RT. The crude product 7 waspurified using preparative RP-HPLC (70 to 80% B in 15 min): t_(R)=8.9min; K′=1.97. Calculated monoisotopic mass for 7 (C₅₄H₇₄N₄O₁₂)=970.53;found: m/z=971.8 [M+H]⁺.

(S)-Fmoc-Asu[(OtBu)KuE(OtBu)₂] (8): For benzyl alcohol (Bzl)deprotection, of 7 (57.2 mg, 65.0 μmol, 1.0 eq.) was dissolved in EtOH(2.0 mL) and palladium on activated charcoal (10%) (5.72 mg, 9.0 μmol,0.1 eq.) was added. The flask was purged beforehand with hydrogen streamand the solution stirred under light hydrogen-pressure (balloon). After70 min stirring, the crude product was filtered through celite, the EtOHevaporated in vacuo and the product purified using preparative RP-HPLC(70 to 70.5% B in 15 min): t_(R)=6.5 min; K′=0.54. Calculatedmonoisotopic mass for 5 (C₄₇H₆₈N₄O₁₂)=880.48; found: m/z=881.8 [M+H]⁺.

OPfp-(S)-Fmoc-Asu[(OtBu)KuE(OtBu)₂] (9):

To a solution of 8 (13.6 mg, 15.4 μmol, 1.0 eq.) in dry DMF was addedDIC (4.77 μL, 1.94 mg, 30.8 μmol, 2.0 eq.) and PfpOH (5.67 mg, 30.8μmol, 2.0 eq.). After 5 min stirring, pyridine (2.49 μL, 31.0 μmol, 2.0eq.) was added and the solution was allowed to stir overnight at RT.Completion of reaction of 9 was analyzed by RP-HPLC (10 to 90% B in 15min): t_(R)=17.2 min; K′=7.6. Calculated monoisotopic mass for 9(C₅₃H₆₇F₅N₄O₁₂)=1046.47; found: m/z=1069.8 [M+Na]⁺.

NHS-2,4-dinitrobenzoate (NHS-DNBA) (27):

To a solution of 2,4-dinitrobenzoic acid (DNBA) (10.0 mg, 47.1 μmol, 1.0eq.) in dry THF was given N, N′-dicyclohexylcarbodiimide (DCC) (9.7 mg,47.1 μmol, 1.0 eq.) and N-hydroxysuccinimide (NHS) (10.8 mg, 94.3 μmol,2.0 eq.) and the reaction mixture was allowed to stir overnight. Thecrude product was purified using RP-HPLC. RP-HPLC (10 to 90% B in 15min): t_(R)=10.21 min K′=4.1. Calculated monoisotopic mass(C₁₁H₇N₃O₈)=309.02; found: not detectable in ESI-MS

DOTAGA-3-iodo-D-Tyr-D-Phe-D-Lys-OH (DOTAGA-y(3-I)fk) (30):

The synthesis of 30 was accomplished via solid phase strategy aspreviously described [2, 3]. In short: The initial starting point wasthe 2-CTC resin loading according to SP-1 of Fmoc-D-Lys(Boc)-OH. Afterconjugation of lysine, Fmoc was deprotected according to SP-3 andFmoc-D-phenylalanine coupled applying SP-2. The same procedure was usedto couple Fmoc-D-Tyr(3-1)-OH. After completion of reaction, the Fmocprotecting group was cleaved according to SP-3 and the resin boundpeptide condensed with the chelator using DOTAGA-anhydride (2.0 eq.) andDIPEA (2.0 eq.) in DMF. The reaction was allowed to stir for 48 h at RT.Finally, the crude product was cleaved from the resin according toSP-7.2 and precipitated in Et₂O and centrifuged. The supernatant wasremoved and 30 purified using RP-HPLC. RP-HPLC (10 to 90% B in 15 min):t_(R)=6.2 min K′=2.1. Calculated monoisotopic mass(C₄₃H₆₁IN₈O₁₄)=1,040.34; found: m/z=1,040.5 [M+H]⁺, m/z=521.3 [M+2H]²⁺,m/z=1,063.4=[M+Na]⁺.

DOTAGA-y(3-I)fk(L-Asu[KuE]) (PSMA-8):

To a solution of DMF containing 30 (5.0 mg, 4.8 μmol, 1.0 eq.), 9 (7.5mg, 7.2 μmol, 1.5 eq.) and DIPEA (3.3 μL, 21.6 μmol, 4.0 eq.) wereadded. The reaction solution was allowed to stir overnight at RT. Aftercompletion of reaction, the solvent was removed in vacuo and the crudeproduct treated with a mixture of piperidine in DMF (20/80; v/v) for 15min to achieve Fmoc-deprotection. The solvent was reduced to approx. 300μL via evaporation in vacuo, precipitated in Et₂O and centrifuged. Withthe resulting pellet was processed according to SP-6 for tBu-removal.The final product was purified via RP-HPLC (10 to 90% B in 15 min):t_(R)=6.09 min K′=2.05. Calculated monoisotopic mass(C₆₃H₉₃IN₁₂O₂₃)=1,512.55; found: m/z=1, 513.9 [M+H]⁺, 757.8 [M+2H]²⁺.

DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) (PSMA-36):

The synthesis of PSMA-36 was achieved by dissolving PSMA-8 (3.0 mg, 3.3μmol, 1.0 eq.) in DMF and addition of 27 (4.1 mg, 13.2 μmol, 4.0 eq.)and DIPEA (2.3 μL, 13.2 μmol, 4.0 eq.). The solution was stirred for 10h at RT and the final product purified by RP-HPLC (10 to 50% B in 15min): t_(R)=12.12 min K′=5.06. Calculated monoisotopic mass(C₇₀H₉₅IN₁₄O₂₈)=1,706.55; found: m/z=1,707.8 [M+H]⁺, 854.7 [M+2H]²⁺.

[^(nat)Lu]DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) ([^(nat)Lu]PSMA-36):RP-HPLC (10 to 60% B in 15 min): t_(R)=9.81 min K′=3.91. Calculatedmonoisotopic mass (C₇₀H₉₂IN₁₄O₂₈Lu)=1,878.47; found: m/z=1,879.9 [M+H]⁺.

Schematic illustration of the synthesis of PSMA-36. (a) HOAt, HATU,DIPEA, benzyl-alcohol, [DMF]; (b) 95% TFA, 5% DCM; (c) 1, HOBt, TBTU,DIPEA, [DMF]; (d) Pd/C (10%), H₂, [EtOH]; (e) DIC, PFP, pyridine, [DMF];(f) 30, DIPEA, [DMF]; (g) 20% piperidine in DMF, [DMF]; (h) TFA; (i) 27,DIPEA [DMF]

4. Synthesis of EuE-Based PSMA Inhibitors PSMA-52 and PSMA-53

DOTAGA-F(4-NO₂)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-52):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with succinic anhydride (4.0 eq.) and DIPEA (1.5eq.) dissolved in DMF. The reaction mixture was allowed to reactovernight at RT. Next, Fmoc-D-Lys-OtBu.HCl (1.5 eq.) was coupledaccording to SP-2 and Fmoc-deprotected as described in SP-3. Thefollowing conjugations with the Fmoc-protected amino acidsFmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Phe(4-NO₂)—OH wereconducted as described in SP-2. The N-terminal Fmoc-deprotected aminoacid was conjugated with the chelator in the final step usingDOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowedto stir for 48 h at RT. After completion of reaction withDOTAGA-anhydride, the peptide was cleaved from the resin according toSP-7.2, the crude product precipitated in Et₂O, centrifuged and thesupernatant removed. The final product was purified via RP-HPLC. RP-HPLC(10 to 60% B in 15 min): t_(R)=9.71 min K′=3.86. Calculated monoisotopicmass (C₇₆H₁₀₀N₁₄O₂₉)=1,672.68; found: m/z=1,673.0 [M+H]⁺.

[^(nat)Lu]DOTAGA-F(4-NO₂)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE)([^(nat)Lu]PSMA-52): RP-HPLC (10 to 60% B in 15 min): t_(R)=9.4 minK′=3.7. Calculated monoisotopic mass (C₇₆H₉₇N₁₄O₂₉Lu)=1,844.6; found:m/z=1,846.0 [M+H]⁺.

2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-53):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with succinic anhydride (4.0 eq.) and DIPEA (1.5eq.) dissolved in DMF. The reaction mixture was allowed to reactovernight at RT. Next, Fmoc-D-Lys-OtBu.HCl (1.5 eq.) was coupledaccording to SP-2 and Fmoc-deprotected as described in SP-3. Thefollowing conjugations with the Fmoc-protected amino acidsFmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and of Fmoc-L-Dap(NHDde)-OH wereconducted as described in SP-2. After coupling of Fmoc-L-Dap(NHDde)-OH,Fmoc-deprotection was achieved as described in SP-3. Next, the freeamino group was conjugated to 2,4-dinitrobenzoic acid (2,4-DNBA) using2,4-DNBA (2.0 eq.), HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (4.0 eq.)in DMF. After completion of reaction, Dde-deprotection was achievedusing SP-5. The N-terminal free amino acid L-Dap was conjugated with thechelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA(2.0 eq.). The reaction was allowed to stir for 48 h at RT. Aftercompletion of reaction with DOTAGA-anhydride, the peptide was cleavedfrom the resin according to SP-7.2, the crude product precipitated inEt₂O, centrifuged and the supernatant removed. The final product waspurified via RP-HPLC.

RP-HPLC (10 to 60% B in 15 min): t_(R)=11.71 min K′=4.86. Calculatedmonoisotopic mass (C₇₇H₁₀₀N₁₆O₃₂)=1,760.67; found: m/z=1,762.1 [M+H]⁺.

[^(nat)Lu]2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE)([^(nat)Lu]PSMA-53): RP-HPLC (10 to 60% B in 15 min): t_(R)=8.3 minK′=3.15. Calculated monoisotopic mass (C₇₇H₉₇N₁₆O₃₂Lu)=1,932.59; found:m/z=1,933.7 [M+H]⁺.

Schematic illustration of the general synthesis procedure of EuE-basedPSMA inhibitors PSMA-52 and PSMA-53 exemplified by PSMA-52. (a) 20%piperidine in DMF, 2, HOBt, TBTU, DIPEA [DMF]; (b) succinic anhydride,DIPEA [DMF]; (c) Fmoc-D/L-Lys-OAII.HCl, HOBt, TBTU, DIPEA [DMF]; (d) 20%piperidine in DMF, Fmoc-D-2-NaI—OH, HOBt, TBTU, DIPEA [DMF]; (e) 20%piperidine in DMF, Fmoc-D-Tyr(OtBu)-OH, HOBt, TBTU, DIPEA [DMF]; (f) 20%piperidine in DMF, Fmoc-D-Phe(4-NH₂)—OH, HOBt, TBTU, DIPEA [DMF]; (g)DOTAGA-anhydride, DIPEA [DMF]; (h) TFA;

5. Synthesis of PSMA-61 and PSMA-62

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-2,4-DNBA) (PSMA-61):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according toSP-2. The amino group of Fmoc-D-Asp-OAII.HCl was deprotected accordingto SP-3 and conjugated to 2,4-DNBA using 2,4-DNBA (1.5 eq.), HOBt (2.0eq.), TBTU (2.0 eq.) and DIPEA (4.0 eq.) in DMF. After completion ofreaction, Allyl-deprotection was achieved according to SP-5. The nextsteps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl (1.5eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Phe(4-NHBoc)-OHaccording to SP-2. The N-terminal Fmoc-deprotected amino acidL-Phe(4-NHBoc)-OH was conjugated with the chelator in the final stepusing DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction wasallowed to stir for 48 h at RT. After completion of reaction withDOTAGA-anhydride, the peptide was cleaved from the resin according toSP-7.2, the crude product precipitated in Et₂O, centrifuged and thesupernatant removed. The final product was purified via RP-HPLC.

RP-HPLC (10 to 90% B in 15 min): t_(R)=6.40 mi K′=2.2. Calculatedmonoisotopic mass (C₈₃H₁₀₅N₁₇O₃₂)=1,851.71; found: m/z=1,852.5 [M+H]⁺,926.7 [M+2H]²⁺.

[^(nat)Lu]DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C4-EuE]-2,4-DNBA)([^(nat)Lu]PSMA-61): RP-HPLC (10 to 90% B in 15 min): t_(R)=8.22 minK′=3.11. Calculated monoisotopic mass (C₈₃H₁₀₂N₁₇O₃₂Lu)=2,023.63; found:m/z=1,013.1 [M+2H]²⁺.

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-62):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according toSP-2. The amino group of Fmoc-D-Asp-OAII.HCl was Fmoc-deprotectedaccording to SP-3 and protected with Dde-OH (2.0 eq.) and DIPEA (4.0eq.) in DMF at RT. The reaction was allowed to stir overnight.Afterwards, Allyl-deprotection of D-Asp was achieved applying SP-5. Thenext steps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl(1.5 eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH andFmoc-L-Phe(4-NHBoc)-OH according to SP-2. In order to conjugate TMA toD-Asp, selective Dde-deprotection was achieved applying SP-4 to affordthe free amino group. TMA was coupled using TMA (2.0 eq.), HOBt (1.5eq.), TBTU (1.5 eq.) and DIPEA (10 eq.) in DMF. The reaction was allowedto stir for 8 h at RT. After conjugation of TMA, Fmoc-deprotection ofFmoc-L-Phe(4-NHBoc)-OH was achieved using SP-3. The N-terminalFmoc-deprotected amino acid L-Phe(4-NHBoc)-OH was conjugated with thechelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA(2.0 eq.). The reaction was allowed to stir for 48 h at RT. Aftercompletion of reaction with DOTAGA-anhydride, the peptide was cleavedfrom the resin according to SP-7.2, the crude product precipitated inEt₂O, centrifuged and the supernatant removed. The final product waspurified via RP-HPLC. RP-HPLC (10 to 70% B in 15 min): t_(R)=7.48 minK′=2.74. Calculated monoisotopic mass (C₈₅H₁₀₇N₁₅O₃₂)=1,849.72; found:m/z=1,850.5 [M+H]*, 925.7 [M+2H]²⁺.

[^(nat)Lu]DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA)([^(nat)Lu]PSMA-62): RP-HPLC (10 to 70% B in 15 min): t_(R)=7.27 minK′=2.64. Calculated monoisotopic mass (C₈₅H₁₀₄N₁₅O₃₂Lu)=2,021.64; found:m/z=1,012.3 [M+2H]²⁺.

6. Synthesis of PSMA-65, PSMA-66 and PSMA-71

2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N⁵-orn-C⁴-EuE) (PSMA-65):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with Fmoc-4-Abz-OH (1.5 eq.), HOAt (1.5 eq.), HATU(1.5 eq.) and DIPEA (4.0 eq.) in DMF. The reaction was allowed to stirovernight at RT. In the next step, the Abz-residue was Fmoc-deprotectedaccording to SP-3. The next steps included the repetitive conjugationwith Fmoc-D-Glu-OtBu, Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH andFmoc-L-Dap(Dde)-OH according to SP-2. After Fmoc-deprotection ofFmoc-L-Dap(Dde)-OH according to SP-3, 2,4-DNBA was coupled using2,4-DNBA (1.5 eq.), HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (4.0 eq.)in DMF. After completion of reaction, the L-Dap(Dde)-residue wasDde-deprotected according to SP-4 and conjugated to the chelator usingDOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowedto stir for 48 h at RT. After completion of reaction withDOTAGA-anhydride, the peptide was cleaved from the resin according toSP-7.2, the crude product precipitated in Et₂O, centrifuged and thesupernatant removed. The final product was purified via RP-HPLC. RP-HPLC(10 to 60% B in 15 min): t_(R)=10.2 min K′=4.1. Calculated monoisotopicmass (C₇₉H₉₆N₁₆O₃₂)=1,780.64; found: m/z=1,781.3 [M+H]⁺.

[^(nat)Lu]2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N⁵-orn-C⁴-EuE)([^(nat)Lu]PSMA-65): RP-HPLC (10 to 60% B in 15 min): t_(R)=9.8 minK′=3.9. Calculated monoisotopic mass (C₇₉H₉₃N₁₆O₃₂Lu)=1,952.56; found:m/z=1.954.0 [M+H]⁺.

DOTAGA-Dap(TMA)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-66):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according toSP-2. The amino group of Fmoc-D-Asp-OAII.HCl was Fmoc-deprotectedaccording to SP-3 and protected with 2.0 eq. Dde-OH and 4.0 eq. DIPEA inDMF. The reaction was allowed to stir overnight. Afterwards,Allyl-deprotection of D-Asp was achieved applying SP-5. The next stepsincluded the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl (1.5 eq.),Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Dap(Dde)-OH according toSP-2. In order to conjugate TMA to D-Asp and L-Dap, selectiveDde-deprotection was achieved applying SP-4 to afford the free aminogroups. TMA was coupled using TMA (4.0 eq.), HOBt (3.0 eq.), TBTU (3.0eq.) and DIPEA (20 eq.) in DMF. The reaction was allowed to stir for 8 hat RT. After conjugation of TMA, Fmoc-deprotection of Fmoc-L-Dap(TMA)-OHwas achieved using SP-3. The N-terminal Fmoc-deprotected amino acidL-Dap was conjugated with the chelator in the final step usingDOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowedto stir for 48 h at RT. After completion of reaction withDOTAGA-anhydride, the peptide was cleaved from the resin according toSP-7.2, the crude product precipitated in Et₂O, centrifuged and thesupernatant removed. The final product was purified via RP-HPLC. RP-HPLC(10 to 70% B in 15 min): t_(R)=7.48 min K′=2.74. Calculated monoisotopicmass (C₈₈H₁₀₇N₁₅O₃₇)=1,965.70; found: m/z=1,966.4 [M+H]⁺, 984.1[M+2H]²⁺.

[^(nat)Lu]DOTAGA-Dap(TMA)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-66)RP-HPLC (10 to 70% B in 15 min): t_(R)=7.46 mi K′=2.73. Calculatedmonoisotopic mass (C₈₈H₁₀₈N₁₅O₃₇Lu)=2,137.62; found: m/z=1,070.4[M+2H]²⁺.

DOTAGA-2-NaI-y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-71):

The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed asdescribed in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step,the Dde-protecting group was cleaved according to SP-4 and the freeamino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according toSP-2. The amino group of Fmoc-D-Asp-OAII.HCl was Fmoc-deprotectedaccording to SP-3 and protected with Dde-OH (2.0 eq.) and DIPEA (4.0eq.) in DMF at RT. The reaction was allowed to stir overnight.Afterwards, Allyl-deprotection of D-Asp was achieved applying SP-5. Thenext steps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl(1.5 eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-2-NaI—OHaccording to SP-2. In order to conjugate TMA to D-Asp, selectiveDde-deprotection was achieved applying SP-4 to afford the free aminogroup. TMA was coupled using TMA (2.0 eq.), HOBt (1.5 eq.), TBTU (1.5eq.) and DIPEA (10 eq.) in DMF. The reaction was allowed to stir for 8 hat RT. After conjugation of TMA, Fmoc-deprotection of Fmoc-L-2-NaI—OHwas achieved using SP-3. The N-terminal Fmoc-deprotected amino acidL-2-NaI—OH was conjugated with the chelator in the final step usingDOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowedto stir for 48 h at RT. After completion of reaction withDOTAGA-anhydride, the peptide was cleaved from the resin according toSP-7.2, the crude product precipitated in Et₂O, centrifuged and thesupernatant removed. The final product was purified via RP-HPLC. RP-HPLC(10 to 80% B in 15 min): t_(R)=7.57 min K′=2.79. Calculated monoisotopicmass (C₈₉H₁₀₈N₁₄O₃₂)=1,884.73; found: m/z=1,886.1 [M+H]⁺, 943.5[M+2H]²⁺.

[^(nat)Lu]DOTAGA-2-NaI-y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA)([^(nat)Lu]PSMA-67): RP-HPLC (10 to 90% B in 15 min): t_(R)=7.81 minK′=2.91. Calculated monoisotopic mass (C₈₉H₁₀₅N₁₄O₃₂Lu)=2,056.64; found:m/z=1,029.7 [M+2H]²⁺.

7. Radiolabeling

⁶⁸Ga-labeling: The ⁶⁸Ge/⁶⁸Ga generator was eluted with aq. HCl (1.0 M),from which a fraction of 1.25 mL, containing approximately 80% of theactivity (600 to 800 MBq), was transferred into a reaction vial(ALLTECH, 5 mL). The vial was beforehand loaded with the respectivecompound (5.0 nmol) and an aq.2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid (HEPES)solution (950 μL, 2.7 M). The reaction vial was heated for 5 min at 95°C. with subsequent fixation of the radiolabeled compound on apreconditioned SPE cartridge (C8 light, SepPak). After purging thecartridge with water (10 mL) in advance, the elution of the radiolabeledPSMA inhibitor from the cartridge was achieved with a mixture of EtOHand water (1/1; v/v), phosphate buffered saline (PBS) (1.0 mL) and againwater (1.0 mL). At the end of radiolabeling, the EtOH was evaporated invacuo and the tracer used without any further purification.Radiochemical purity was controlled using Radio-TLC (1.0 M sodiumcitrate buffer and 0.06 M NH₄OAc/MeOH buffer (1/1; v/v)).

¹⁷⁷Lu-labeling: The ⁷⁷Lu-labeled compounds were prepared as previouslydescribed [5] with minor modifications and used without furtherpurification. In short, to NH₄OAc-buffer (10 μL, 1.0 M, pH=5.9) wasadded the respective tracer (0.75 to 1.0 nmol, 7.5 to 10 μL), ¹⁷⁷LuCl₃(10 to 40 MBq; A_(S)>3000 GBq/mg, 740 MBq/mL, 0.04 M HCl, ITG, Garching,Germany) and finally filled with trace-pure water (up to 100 μL) (Merck,Darmstadt, Germany). The reaction mixture was heated for 40 min at 95°C. and the radiochemical purity was determined using radio-TLC.

¹²⁵I-labeling: Briefly, the stannylated precursor(SnBu₃-BA)(OtBu)KuE(OtBu)₂ (PSMA-45) (approx. 0.1 mg) was dissolved in asolution containing peracetic acid (20 μL), [¹²⁵]NaI (5.0 μL, approx.21.0 MBq) (74 TBq/mmol, 3.1 GBq/mL, 40 mM NaOH, Hartmann Analytic,Braunschweig, Germany), MeCN (20 μL) and AcOH (10 μL). The reactionsolution was incubated for 10 min at RT, loaded on a cartridge (C18 SepPak Plus, preconditioned with 10 mL MeCOH and 10 mL water) and rinsedwith water (10 mL). After elution with a 1/1 mix (v/v) of EtOH and MeCN(2.0 mL), the solution was evaporated to dryness under a gentle nitrogenstream and treated with TFA (200 μL) for 30 min with subsequentevaporation of TFA. The crude product of ([¹²⁵I]I-BA)KuE was purified byradio-RP-HPLC (20 to 40% B in 20 min): t_(R)=13.0 min; K′=6.2.

8. Determination of HSA Binding

HSA binding experiments were performed as previously described [6]. Themobile phase consisted of a binary gradient system with a constant totalflow rate of 0.5 mL/min. Mobile phase A was a 50 mm pH 6.9NH₄OAc-solution, mobile phase B was 2-Propanol (RP-HPLC grade, VWR,Germany). The gradient of mobile phase A was 100% from 0 to 3 min andfrom 3 min to the end of each run mobile phase B was set 20%. At eachexperimental day, the column was calibrated with nine referencesubstances to confirm the performance and to establish the non-linearregression. PSMA inhibitors were dissolved in a 0.5 mg/mL concentrationin a mixture of 2-Propanol and NH₄OAc-buffer (50 mm pH 6.9) (1/1; v/v).For each run, 10 μL of the solution containing the inhibitor wasinjected into the RP-HPLC system and the retention time measured. Theliterature HSA binding [%] was obtained from Valko et. al. or Yamazakiet al. [6, 7]. Non-linear regression was established with OriginPro2016G.

9. Determination of Lipophilicity

Lipophilicity: The radiolabeled PSMA inhibitor (0.5 to 1.0 MBq)dissolved in PBS (500 μL, pH=7.4), was added to n-octanol (500 μL) in areaction vial (1.5 mL), which was rigorously vortexed for 3 min (n=6).For quantitative phase separation, the mixture was centrifuged at 6,000g for 5 min (Biofuge 15, Heraus Sepatech, Osterode, Germany). Theactivity from samples of each phase (100 μL) were measured in aγ-counter to obtain the log P_((o/w)) value.

10. Cell Experiments

Cell culture: PSMA-positive LNCAP cells (300265; Cell Lines ServiceGmbH) were cultivated in Dulbecco modified Eagle medium/NutritionMixture F-12 (1/1) (DMEM-F12, Biochrom) supplemented with fetal calfserum (FCS) (10%, Biochrom) and kept at 37° C. in a humidified CO₂atmosphere (5%). One day (24 h±2 h) prior to all experiments with LNCaPcells, the cultivated cells were harvested using a mixture oftrypsin/ethylendiaminetetraacetate (0.05%/0.02%) and PBS andcentrifuged. After centrifugation, the supernatant was disposed and thecell pellet resuspended in culture medium. Afterwards, cells werecounted with a hemocytometer (Neubauer) and seeded in 24-well plates.IC₅₀ values were determined transferring 150,000 cells/mL per well into24-well plates, whereas internalization rates were obtained bytransferring 125,000 cells/mL per well into 24-well PLL-coated plates.

11. Affinity (IC₅₀)

After removal of the culture medium, the cells were treated once withHBSS (500 μL, Hank's balanced salt solution, Biochrom, Berlin, Germany,with addition of 1% BSA) and left 15 min on ice for equilibration inHBSS (200 μL, 1% BSA). Next, solutions (25 μL per well) containingeither HBSS (1% BSA, control) or the respective ligand in increasingconcentration (10⁻¹⁰ to 10⁻⁴ m in HBSS (1% BSA)) were added withsubsequent addition of ([¹²⁵I]I-BA)KuE (25 μL, 2.0 nm) in HBSS (1% BSA).All experiments were performed at least three times for eachconcentration. After 60 min incubation on ice, the experiment wasterminated by removal of the medium and consecutive rinsing with HBSS(200 μL). The media of both steps were combined in one fraction andrepresent the amount of free radioligand. Afterwards, the cells werelysed with NaOH (250 μL, 1.0 m) and united with the HBSS (200 μL) of thefollowing washing step. Quantification of bound and free radioligand wasaccomplished in a γ-counter.

12. Internalization

Subsequent to the removal of the culture medium, the cells were washedonce with DMEM-F12-solution (500 μL, 5% BSA) and left to equilibrate forat least 15 min at 37° C. in DMEM-F12-solution (200 μL, 5% BSA).Afterwards, each well was treated with either DMEM-F12-solution (25 μL,5% BSA) or 2-PMPA-solution (25 μL, 100 μm) for blockade. Next, therespective ⁶⁸Ga- or ¹⁷⁷Lu-labeled PSMA inhibitor (25 μL; 2.0 nm and 10nm, respectively) was added and the cells incubated at 37° C. for 5, 15,30 and 60 min, respectively. The experiment was terminated by placingthe 24-well plate on ice for 3 min and the consecutive removal of themedium. Each well was rinsed with HBSS (250 μL) and the fractions fromthese first two steps combined, representing the amount of freeradioligand. Removal of surface bound activity was accomplished byincubation of the cells with ice-cold 2-PMPA-solution (250 μL, 10 μm inPBS) for 5 min and subsequent rinsing with ice-cold PBS (250 μL). Theinternalized activity was determined through incubation of the cells inNaOH (250 μL, 1.0 m) and the combination with the fraction of thesubsequent washing step with again NaOH (250 μL, 1.0 m). Each experiment(control and blockade) was performed in triplicate for each time point.Free, surface bound and internalized activity was quantified in aγ-counter.

13. Externalization

Externalization kinetics of the radiolabeled PSMA inhibitors weredetermined using LNCaP cells, which were similarly prepared as describedfor the internalization assay. After an initial cell-washing step withDMEM-F12-solution (5% BSA), the cells were left to recondition for atleast 15 min at 37° C. Subsequently, the LNCaP cells were incubated withthe respective radiolabeled peptide (25 μL, 10.0 nm) at 37° C. for 60min in a total volume of 250 μL in each well. After 60 min, thesupernatant with the unbound free fraction was removed and measured in aγ-counter for the calculation of total added radioactivity. An acid washstep was avoided to warrant enzyme integrity during the followingexternalization and recycling study. To determine the recycling rate,fresh DMEM-F12-solution (250 μL, 5% BSA) was given to the cells to allowre-internalization. In contrast, re-internalization was inhibited byaddition of DMEM-F12-solution containing 2-PMPA (225 μL DMEM-F12 (5%BSA) and 25 μL of 100 μm 2-PMPA-solution (PBS)). The cells were thenincubated for 0, 20, 40 and 60 min at 37° C. Consequently, thesupernatant was removed and the cells were washed with ice-cold HBSS(250 μL). The combination of the supernatant and the volume of theconcomitant washing step with HBSS (200 μL) account for externalizedradioligand at the investigated time point. Further, the cells were thenwashed with ice-cold 2-PMPA HBSS solution (250 μL, 10 μm) twice,combined and thus represented the fraction of membrane-boundradioligand. The determination of the internalized fraction was achievedby lysis as described for the internalization assay with NaOH (250 μL,1.0 m). The activities of free, externalized, membrane-bound andinternalized radioligand were quantified in a γ-counter.

14. Animal Experiments

All animal experiments were carried out in accordance with the generalanimal welfare regulations in Germany (Deutsches Tierschutzgesetz,approval #55.2-1-54-2532-71-13). For the tumor model, LNCaP cells(approx. 10⁷ cells) were suspended in serum-free DMEM-F12 medium andMatrigel (1/1; v/v) (BD Biosciences, Germany) and inoculated onto theright shoulder of male, 6 to 8 weeks old CB-17 SCID mice (Charles RiverLaboratories, Sulzfeld, Germany). Animals were used after the tumor sizereached 4 to 8 mm in diameter for experiments.

15. PET

Imaging experiments were conducted using a Siemens Inveon small animalPET and the data analyzed by the associated Inveon Research Workplacesoftware. Mice were anaesthetized with isoflurane and approx. 4.0 to 17MBq of the ⁶⁸Ga-labeled compounds were injected via tail vein (approx.150 to 300 μL). Dynamic imaging was carried out after on-bed injectionfor 90 min. The static blockade image was obtained after 1 h p.i. with15 min acquisition time. PSMA-blockade was achieved by coinjection of 8mg/kg of 2-PMPA-solution (PBS). All images were reconstructed using anOSEM3D algorithm without scanner and attenuation correction.

16. Biodistribution

Approximately 4.0 to 12.0 MBq (approx. 150 to 300 μL) of the respective⁶⁸Ga- or ¹⁷⁷Lu-labeled PSMA inhibitors were injected into the tail veinof LNCaP tumor-bearing male CB-17 SCID mice, which were sacrificed aftera specific timeframe (n=4, respectively). Selected organs were removed,weighted and measured in a γ-counter.

17. References in Example 1

-   1. Šimeček, J., et al., A Monoreactive Bifunctional    Triazacyclononane Phosphinate Chelator with High Selectivity for    Gallium-68. Chem Med Chem, 2012. 7(8): p. 1375-1378.-   2. Weineisen, M., et al., Development and first in human evaluation    of PSMA I&T—A ligand for diagnostic imaging and endoradiotherapy of    prostate cancer. Journal of Nuclear Medicine, 2014. 55(supplement    1): p. 1083-1083.-   3. Weineisen, M., et al., Synthesis and preclinical evaluation of    DOTAGA-conjugated PSMA ligands for functional imaging and    endoradiotherapy of prostate cancer. EJNMMI research, 2014. 4(1): p.    1.-   4. Weineisen, M., et al., 68Ga- and 177Lu-Labeled PSMA I&T:    Optimization of a PSMA-Targeted Theranostic Concept and First    Proof-of-Concept Human Studies. Journal of Nuclear Medicine, 2015.    56(8): p. 1169-1176.-   5. Sosabowski, J. K. and S. J. Mather, Conjugation of DOTA-like    chelating agents to peptides and radiolabeling with trivalent    metallic isotopes. Nat. Protocols, 2006. 1(2): p. 972-976.-   6. Valko, K., et al., Fast gradient HPLC method to determine    compounds binding to human serum albumin. Relationships with    octanol/water and immobilized artificial membrane lipophilicity.    Journal of pharmaceutical sciences, 2003. 92(11): p. 2236-2248.-   7. Yamazaki, K. and M. Kanaoka, Computational prediction of the    plasma protein-binding percent of diverse pharmaceutical compounds.    Journal of pharmaceutical sciences, 2004. 93(6): p. 1480-1494.

EXAMPLE 2: RESULTS 1. Effect of the Introduction of 2,4-DinitrobenzoicAcid into the Linker Area of PSMA I&T

DOTAGA-y(3-I)fk(Sub-KuE) (PSMA I&T):

DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) (PSMA-36):

TABLE 3 IC₅₀ Internalization HSA PSMA Inhibitor Configuration [nM] [%]logP [%] [^(nat/177)Lu]PSMA I&T —  7.9 ± 2.4* 75.5 ± 1.6* −4.12 ± 0.11*78.6 [^(nat/177)Lu]PSMA-36 2,4-DNBA- 5.3 ± 1.0 189.8 ± 37.5  n.d. 82.5→ Slightly higher affinity and increase of internalization by 251%.

2. The Binding Motif was Changed from EuK to EuE and the Peptide Spacerfrom -y(3-I)fk- to -y-2-naI-k-

DOTAGA-y(3-I)fk(Sub-KuE) (PSMA I&T):

DOTAGA-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-46):

TABLE 4 IC₅₀ Internalization HSA PSMA inhibitor Configuration [nM] [%]logP [%] [^(nat/177)Lu]PSMA I&T -y(3-I)fk- ∥ -KuE 7.9 ± 2.4  75.5 ± 1.6−4.12 ± 0.11 78.6 [^(nat/177)Lu]PSMA-46 -y-2-nal-k- ∥ -EuE 3.2 ± 1.1216.2 ± 9.2 −4.21 ± 0.08 57.7 → Compared to the reference PSMA I&T, theimproved reference compound PSMA-46 showed higher internalization andexhibited improved affinity. Thus, based on the structure PSMA-46,electron deficient aromatic residues were introduced in a furtherdevelopment step.

3. 4-Nitrophenylalanine and 2,4-DNBA were Introduced into the PeptideSpacer of PSMA-46

DOTAGA-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-46):

DOTAGA-F(4-NO₂)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-52):

2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N⁵-orn-C⁴-EuE) (PSMA-53):

TABLE 5 IC₅₀ Internalization HSA PSMA inhibitor Configuration [nM] [%]logP [%] [^(nat/177)Lu]PSMA-46 -y-2-nal-k- ∥ -EuE 3.2 ± 1.1 216.2 ± 9.2−4.21 ± 0.08 57.7 [^(nat/177)Lu]PSMA-52 -F(4-NO₂)y-2-nal-k- 3.4 ± 0.2229.9 ± 8.0 −4.11 ± 0.07 95.4 [^(nat/177)Lu]PSMA-53 2,4 DNBA-Dap-y-2-3.2 ± 0.5  293.6 ± 10.0 −4.08 ± 0.04 95.9 nal-k- → While the affinityremained similar, the introduction of 4-nitrophenylalanine increasedslightly the internalization, however, by introduction of a furthernitro group through 2,4-DNBA, a significant increase of internalizationwas possible. → Two electron withdrawing groups are preferred in orderto increase the internalization.

4. Introduction of 4-amino-phenylalanine

DOTAGA-F(4-NH₂)y-2-naI-k(Suc-N⁵-orn-C4-EuE) (PSMA-49):

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-2,4-DNBA) (PSMA-61):

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-62):

TABLE 6 IC₅₀ Internalization HSA PSMA inhibitor Configuration [nM] [%]logP [%] [^(nat/177)Lu]PSMA I&T -k(Sub-ε-KuE) 7.9 ± 2.4  75.5 ± 1.6−4.12 ± 0.11 78.6 [^(nat/177)Lu]PSMA-49 -k(Suc-δ-orn(γ-EuE)) 2.5 ± 0.6245.0 ± 4.2 −4.01 ± 0.11 74.2 [^(nat/177)Lu]PSMA-61 -k((2,4-DNBA)-d-δ-4.5 ± 0.4  359.5 ± 22.6 −4.07 ± 0.05 63.3 orn(γ-EuE))[^(nat/177)Lu]PSMA-62 -k((TMA)-d-δ-orn(γ- 4.0 ± 0.2 343.9 ± 6.0 −4.12 ±0.05 >91.0 EuE)) → Both modifications, 2,4-DNBA and Trimesic acid, wereable to further increase the internalization.

5. Introduction of an Electron Deficient Group in the Peptide Spacer

DOTAGA-F(4-NH₂)y-2-naI-e(Abz-N⁵-orn-C⁴-EuE) (PSMA-60):

2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N⁵-orn-C⁴-EuE) (PSMA-65):

TABLE 7 IC₅₀ Internalization HSA PSMA inhibitor Configuration [nM] [%]logP [%] [^(nat/177)Lu]PSMA-60 -e(Abz-δ-orn(γ-EuE)) 6.6 ± 1.5 267.4 ±7.9  −3.85 ± 0.13 98.5 [^(nat/177)Lu]PSMA-65 2,4-DNBA-Dap 3.5 ± 0.3340.2 ± 18.9 −4.15 ± 0.08 98.7 (DOTAGA)-y-2-nal- e(Abz-[HO-δ-orn-[γ-EuE]])-OH → The electron deficient aromatic modification 2,4-DNBA wasable to increase the internalization.

6. Trimesic Acid was Incorporated into the Linker and Peptide Spacer ofthe PSMA Inhibitors

DOTAGA-F(4-NH₂)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-62):

DOTAGA-Dap(TMA)y-2-naI-k(d[N⁵-orn-C⁴-EuE]-TMA) (PSMA-66):

TABLE 8 IC₅₀ Internalization HSA PSMA inhibitor Configuration [nM] [%]logP [%] [^(nat/177)Lu]PSMA I&T -k(Sub-ε-KuE)  7.9 ± 2.4*  75.5 ± 1.6* −4.12 ± 0.11* 78.6 [^(nat/177)Lu]PSMA-62 -k((TMA)-d-δ-orn(γ- 4.0 ± 0.2343.9 ± 6.0 −4.12 ± 0.05 >91.0 EuE)) [^(nat/177)Lu]PSMA-66 DOTAGA- 3.8 ±0.3 297.8 ± 2.0 −4.25 ± 0.14 64.4 Dap(TMA)y-2-nal- k(d[N5-orn-C4-EuE]-TMA) → The exchange of 4-amino-phenylalanine to Dap(TMA) resultedin similar affinity but a slightly reduced internalization capacity.Since both ligands seemed highly promising, both tracer were evaluatedin further experiments. → The conclusion from these experiments is thatan electron deficient aromatic residue is transferable and able toincrease the internalization while maintaining a high affinity.

7. The Influence of Internalization on the Cell Retention In Vitro wasEvaluated for the Compounds [¹⁷⁷Lu]PSMA-62 and [¹⁷⁷Lu]PSMA-66 inComparison to [¹⁷⁷Lu]PSMA I&T and [¹⁷⁷Lu]PSMA-617

[¹⁷⁷Lu]PSMA-66 demonstrated the highest intracellular activity in thetumor cells after 1 h followed by [¹⁷⁷Lu]PSMA-62, although theinternalization of [¹⁷⁷Lu]PSMA-62 was found to be higher than for[¹⁷⁷Lu]PSMA-66 (343.9% vs. 297.8%; respectively). Interestingly, evenwhen re-internalization was blocked with 100 μM 2-PMPA-solution, theintracellular clearance [¹⁷⁷Lu]PSMA-66 was lower than for all otherinvestigated compounds. The difference compared to reference [¹¹⁷Lu]PSMAI&T was more than twofold, if re-internalization was blocked.

[¹⁷⁷Lu]PSMA-66 has nine free carboxylic groups, which equal ninenegative charges in vivo (pH=7.4). The extensively charged character ofthis compound could be a possible explanation for the protractedintracellular retention due to electrostatic repulsive effects from thenegatively charged cell membranes.

8. In Vivo Experiments: Biodistribution

TABLE 9 Biodistribution data of [¹⁷⁷Lu]PSMA-49, [¹⁷⁷Lu]PSMA-62 and[¹⁷⁷Lu]PSMA-66 (in % ID/g) in LNCaP-tumor xenograft bearing CB-17 SCIDmice at 1 h p.i. (n = 4, respectively). Between 3.5 MBq and 5.5 MBq ofthe respective ¹⁷⁷Lu-labeled radioligand were injected (0.15 to 0.25nmol tracer). [¹⁷⁷Lu]PSMA I&T [¹⁷⁷Lu]PSMA-49 [¹⁷⁷Lu]PSMA-62[¹⁷⁷Lu]PSMA-66 1 h p.i. 1 h p.i. 1 h p.i. 1 h p.i. blood 0.37 ± 0.100.46 ± 0.06 0.52 ± 0.03 0.50 ± 0.03 heart 0.58 ± 0.15 0.57 ± 0.11 0.58 ±0.06 0.49 ± 0.06 lung 1.32 ± 0.45 1.47 ± 0.25 0.87 ± 0.11 0.62 ± 0.12liver 0.37 ± 0.10 0.99 ± 0.19 0.37 ± 0.04 0.32 ± 0.02 spleen 13.8 ± 4.5920.53 ± 6.79  4.62 ± 1.81 1.93 ± 0.04 pancreas 1.30 ± 1.39 0.56 ± 0.070.18 ± 0.05 0.15 ± 0.04 stomach 0.29 ± 0.06 0.35 ± 0.10 0.51 ± 0.2  0.28± 0.04 intestine 0.59 ± 0.50 0.30 ± 0.08 0.49 ± 0.25 0.21 ± 0.03 kidney128.90 ± 10.74  162.96 ± 23.20  106.45 ± 17.18  117.47 ± 6.86  adrenalgland 6.25 ± 2.59 5.66 ± 1.66 1.92 ± 0.80 0.78 ± 0.07 muscle 0.18 ± 0.070.17 ± 0.02 0.12 ± 0.03 0.19 ± 0.07 bone 0.14 ± 0.04 0.29 ± 0.14 0.30 ±0.08 0.37 ± 0.13 brain 0.03 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 0.02 ± 0.01tumor 4.69 ± 0.95 8.21 ± 0.23 8.00 ± 0.78 10.00 ± 0.44  tumor/blood 12.717.8 15.4 20.0 tumor/kidney  0.04  0.05  0.08  0.09 tumor/muscle 26.148.3 66.7 52.6

Compared to the tumor uptake of [¹⁷⁷Lu]PSMA I&T after 1 h p.i.(4.69±0.95%), a significant increase in tumor activity was achievedthrough the improvement of internalization and affinity. As alreadyobserved for [¹⁷⁷Lu]PSMA-16, [¹⁷⁷Lu]PSMA-40 and [¹⁷⁷Lu]PSMA-41, theextension of the peptide spacer with 4-amino-D-phenylalanine led to highkidney uptake and confirmed that this modification increases renalaccumulation.

The introduction of trimesic acid into the linker of [¹⁷⁷Lu]PSMA-62 ledto a reduction of renal uptake (106.45±17.18% vs. 162.96±23.20%,respectively) and slightly lower tumor uptake compared to the reference.Since the internalization for [¹⁷⁷Lu]PSMA-62 was higher in directcomparison to [¹⁷⁷Lu]PSMA-49, the lower tumor uptake was unexpected. Itis unclear to what extent internalization contributes to tumor uptakeand if it is less important than affinity. The direct comparison of[¹⁷⁷Lu]PSMA-49 and [¹⁷⁷Lu]PSMA-62 indicates that affinity is morecrucial since [¹⁷⁷Lu]PSMA-49 was more affine towards PSMA (2.5±0.6 nMvs. 4.0±0.2 nM, respectively).

TABLE 10 Biodistribution data of [¹⁷⁷Lu]PSMA I&T, [¹⁷⁷Lu]PSMA-62 and[¹⁷⁷Lu]PSMA- 66 and [¹⁷⁷Lu]PSMA-71 (in % ID/g) in LNCaP-tumor xenograftbearing CB-17 SCID mice after 24 h p.i. (n = 4, respectively). Between3.5 MBq and 5.5 MBq of the respective ¹⁷⁷Lu-labeled radioligand wereinjected (0.15 to 0.25 nmol tracer). [¹⁷⁷Lu]PSMA I&T [¹⁷⁷Lu]PSMA-62[¹⁷⁷Lu]PSMA-66 [¹⁷⁷Lu]PSMA-71 24 h p.i. 24 h p.i. 24 h p.i. * 24 h p.i.blood 0.012 ± 0.01  0.004 ± 0.001 0.003 ± 0.001 0.008 ± 0.001 heart 0.05± 0.03 0.02 ± 0.01  0.03 ± 0.007 0.07 ± 0.01 lung 0.16 ± 0.03 0.03 ±0.02  0.03 ± 0.007 0.05 ± 0.01 liver 0.05 ± 0.01 0.17 ± 0.07 0.14 ± 0.020.38 ± 0.14 spleen 1.94 ± 1.01 0.09 ± 0.06 0.09 ± 0.02 0.27 ± 0.15pancreas 0.05 ± 0.02  0.01 ± 0.002  0.02 ± 0.007 0.15 ± 0.15 stomach0.05 ± 0.02 0.03 ± 0.01 0.07 ± 0.03 0.20 ± 0.10 intestine 0.12 ± 0.060.03 ± 0.02 0.11 ± 0.07 0.27 ± 015  kidney 34.66 ± 17.20 5.26 ± 1.5820.92 ± 2.51  32.36 ± 2.49  adrenal g. 1.06 ± 0.24 0.04 ± 0.02 0.09 ±0.09 0.32 ± 0.15 muscle 0.01 ± 0.01 0.007 ± 0.001  0.02 ± 0.006  0.01 ±0.001 bone 0.01 ± 0.01  0.04 ± 0.006 0.02 ± 0.01 0.04 ± 0.02 brain 0.02± 0.01  0.01 ± 0.006  0.01 ± 0.001  0.01 ± 0.002 tumor 4.06 ± 1.12 7.70± 1.35 5.73 ± 1.39 14.29 ± 0.89  t/blood 406 1925.0 1910.0 1786.3t/kidney 0.1 1.5 0.3 0.4 t/muscle 406 1100.0 286.5 1429.0 t/liver 81.245.3 40.9 37.6

The results in Table 10 show distinct differences between the evaluatedtracer [¹⁷⁷Lu]PSMA-62, [¹⁷⁷Lu]PSMA-66 and [¹⁷⁷Lu]PSMA-71. Regardingrenal clearance, it was visible that for all ligands a decrease in renaluptake compared to 1 h p.i. (Table 9) was observed. While [¹⁷⁷Lu]PSMAI&T demonstrated after 24 h p.i. the highest renal uptake,[¹⁷⁷Lu]PSMA-62 showed the lowest, which is in concordance to theobserved renal clearance in the PET-study. The tumor uptake of[¹⁷⁷Lu]PSMA-61 after 24 h p.i. remained almost stable over 23 h(8.00±0.75 vs. 7.70±1.35% ID/g, 1 h p.i. and 24 h p.i., respectively).Although [¹⁷⁷Lu]PSMA-62 and [¹⁷⁷Lu]PSMA-66 demonstrated similar in vitroparameter regarding internalization and affinity, the tumor uptake of[¹⁷⁷Lu]PSMA-66 decreased to a greater extent from 1 h p.i. to 24 h p.i.(10.00±0.44 vs. 5.73±1.39% ID/g, 1 h p.i. and 24 h p.i., respectively)compared to [¹⁷⁷Lu]PSMA-62. The stronger tumor retention together withthe more beneficial tumor to liver and tumor to muscle ratios render[¹⁷⁷Lu]PSMA-62 superior compared to [¹⁷⁷Lu]PSMA-66. The highest tumoruptake was observed for PSMA-71, which also exhibited the highestHSA-binding value. While the kidney uptake 24 h p.i. was similar to[¹⁷⁷Lu]PSMA I&T, the tumor uptake was more than threefold higher for[¹⁷⁷Lu]PSMA-71 (4.06±1.12 vs. 14.29±0.89% ID/g, [¹⁷⁷Lu]PSMA I&T and[¹⁷⁷Lu]PSMA-71, respectively).

In this respect, [¹⁷⁷Lu]PSMA-71 may be considered as a particularlyvaluable tracer for endoradiotherapeutic application and is a candidatefor clinical application.

9. In Vivo Experiments: PET-Imaging

Effect of 2,4-Dinitrobenzoic Linker Substitution on EuK-Based Inhibitors

The EuK-based inhibitor PSMA-36 was evaluated in a small animal PET scanto examine the influence of the 2,4-dinitrobenzoic acid in the linker onthe in vivo distribution.

The logarithmic TACs plot shows specific kidney and tumor uptake of[⁶⁸Ga]PSMA-36. Linear decrease of the blood pool activity and in themuscle region imply low unspecific binding and fast excretion.Accumulation in the tumor remained steady over the observed period.Although [¹⁷⁷Lu]PSMA-36 exhibited a more than threefold higherinternalization rate than [¹⁷⁷Lu]PSMA I&T, tumor uptake was onlymoderate with 3.5% ID/mL after 85 min p.i. The most significantdifference compared to [⁶⁸Ga]PSMA I&T was the high and steady uptake inthe lacrimal and salivary gland, displaying approx. 2% ID/mL in bothregions. Since the only structural difference to the reference[⁶⁸Ga]PSMA I&T is the introduction of 2,4-dinitrobenzoic acid, thelinker modification must be the reason for this enhanced uptake.However, further studies are necessary to confirm this effect.

It is also interesting, that the clearance in these regions was slowercompared to the blood pool and muscle, which implies that a distinctretaining mechanism is involved. It was reported that PSMA participatesin angiogenesis during ocular neovascularization in mice and mighttherefore explain the uptake of [⁶⁸Ga]PSMA-36 [1]. Tracer accumulationin the salivary glands is a common problem during clinical therapeuticapproaches with ¹⁷⁷Lu-labeled PSMA inhibitors [2]. Drug uptake into thesalivary glands depends on intra- or extracellular pathways and mostcommonly on simple diffusion among the phospholipid bilayer of theacinar cells. Saliva drug concentrations are reflected predominantly bythe free, non-ionized fraction in the blood plasma regarding passivediffusion [3-5]. In this respect, it seems highly unlikely that passivediffusion is responsible for the salivary gland uptake. Other mechanismshave to be involved since EuK-based PSMA inhibitors are highly chargedin vivo and thus exhibit high polarity. Further, passive diffusion wouldbe visualized during PET scans in every region as high backgroundactivity, which does not occur for most PSMA ligands since the rapidclearance removes the tracer from the blood pool.

Effect of Trimesic Acid on EuE-Based Inhibitors

Substitution of the PSMA ligands with electron deficient aromaticsystems resulted in enhanced internalization rates of [¹⁷⁷Lu]PSMA-62 and[¹⁷⁷Lu]PSMA-66 (343.9% and 297.8%, respectively). Both ligands weretherefore evaluated and compared among each other in PET studies.

Both tracer exhibited excellent tracer kinetics regarding kidney, muscleand blood pool uptake. Specific uptake in the kidneys was slightlyhigher for [⁶⁸Ga]PSMA-66 compared to [⁶⁸Ga]PSMA-62 (45.3% ID/mL vs.34.8% ID/mL, respectively). The higher renal accumulation in the PETscan of [⁶⁸Ga]PSMA-66 compared to [⁶⁸Ga]PSMA-62 nicely correlated withthe biodistribution experiments. TACs for muscle and blood pool activityshowed linear uptake and ongoing clearance from these compartments.

10. References in Example 2

-   1. Grant, C. L., et al., Prostate specific membrane antigen (PSMA)    regulates angiogenesis independently of VEGF during ocular    neovascularization. PloS one, 2012. 7(7): p. e41285.-   2. Kulkarni, H. R., et al., PSMA-Based Radioligand Therapy for    Metastatic Castration-Resistant Prostate Cancer: The Bad Berka    Experience Since 2013. Journal of Nuclear Medicine, 2016.    57(Supplement 3): p. 97S-104S.-   3. Haeckel, R., Factors influencing the saliva/plasma ratio of    drugs. Annals of the New York Academy of Sciences, 1993. 694(1): p.    128-142.-   4. Jusko, W. J. and R. L. Milsap, Pharmacokinetic Principles of Drug    Distribution in Salivaa. Annals of the New York Academy of    Sciences, 1993. 694(1): p. 36-47.-   5. Aps, J. K. and L. C. Martens, Review: the physiology of saliva    and transfer of drugs into saliva. Forensic science    international, 2005. 150(2): p. 119-131.-   6. Young, J. D., et al., 68Ga-THP-PSMA: a PET imaging agent for    prostate cancer offering rapid, room temperature, one-step kit-based    radiolabeling. Journal of Nuclear Medicine, 2017: p. jnumed.    117.191882.-   7. Wüstemann, T., et al., Design of Internalizing PSMA-specific    Glu-ureido-based Radiotherapeuticals. Theranostics, 2016. 6(8): p.    1085.-   8. Hao, G., et al., A multivalent approach of imaging probe design    to overcome an endogenous anion binding competition for noninvasive    assessment of prostate specific membrane antigen. Molecular    pharmaceutics, 2013. 10(8): p. 2975-2985.-   9. Soret, M., S. L. Bacharach, and I. Buvat, Partial-volume effect    in PET tumor imaging. Journal of Nuclear Medicine, 2007. 48(6): p.    932-945.-   10. Bao, Q., et al., Performance evaluation of the inveon dedicated    PET preclinical tomograph based on the NEMA NU-4 standards. Journal    of Nuclear Medicine, 2009. 50(3): p. 401-408.

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,

wherein: m is an integer of 2 to 6; n is an integer of 2 to 6; R^(1L) is CH₂, NH or O; R^(2L) is C or P(OH); R^(3L) is CH₂, NH or O; X¹ is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, and an amine bond; L¹ is a divalent linking group with a structure selected from an oligoamide, an oligoether, an oligothioether, an oligoester, an oligothioester, an oligourea, an oligo(ether-amide), an oligo(thioether-amide), an oligo(ester-amide), an oligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), an oligo(ether-ester), an oligo(ether-thioester), an oligo(ether-urea), an oligo(thioether-ester), an oligo(thioether-thioester), an oligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea), and an oligo(thioester-urea), which linking group may carry a group EDS; X² is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, and an amine bond; R² is an optionally substituted aryl group or an optionally substituted aralkyl group, which aryl group or aralkyl group may be substituted on its aromatic ring with one or more substituents selected from halogen and —OH; R³ is an optionally substituted aryl group or an optionally substituted aralkyl group, which aryl group or aralkyl group may be substituted on its aromatic ring with one or more substituents selected from halogen and —OH; r is 0 or 1; p is 0 or 1; q is 0 or 1; R⁴ is selected from an optionally substituted aryl group and a group EDS, which aryl group may be substituted on its aromatic ring with one or more substituents selected from halogen, —OH and —NH₂; X³ is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, an amine bond, and a group of the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) and the other marked bond attaches X³ to the remainder of the compound of formula (I); R^(M) is a marker group which comprises a chelating group optionally containing a chelated non-radioactive or radioactive cation; and wherein the group EDS is contained at least once in the compound of formula (I) and has a structure selected from (E-1A), (E-1B), (E-2A) and (E-2B):

wherein

marks the bond which attaches the group EDS, to the remainder of the compound of formula (I); s is 1, 2 or 3, preferably 1 or 2, and more preferably 1; t is 1, 2 or 3, preferably 1 or 2, and more preferably 2; R^(5A) is, independently for each occurrence for s>1, an electron withdrawing substituent, which is preferably selected from —NO₂ and —COOH, and which is more preferably —COOH, and wherein the bond between R^(5A) and the phenyl ring indicates that the s groups R^(5A) replace s hydrogen atoms at any position on the phenyl ring; R^(5B) is, independently for each occurrence for s>1, a substituent carrying an electron lone pair at the atom directly attached to the phenyl ring shown in formula (E-1B), which substituent is preferably selected from —OH and —NH₂, and which is more preferably —NH₂, and wherein the bond between R^(5B) and the phenyl ring indicates that the s groups R^(5B) replace s hydrogen atoms at any position on the phenyl ring; R^(6A) is, independently for each occurrence for t>1, an electron withdrawing substituent, which is preferably selected from —NO₂ and —COOH, and which is more preferably —COOH, and wherein the bond between R^(6A) and the phenyl ring indicates that the t groups R^(6A) replace t hydrogen atoms at any position on the phenyl ring; and R^(6B) is, independently for each occurrence for t>1, a substituent carrying an electron lone pair at the atom directly attached to the phenyl ring shown in formula (E-1B), which substituent is preferably selected from —OH and —NH₂, and which is more preferably —OH, and wherein the bond between R^(6B) and the phenyl ring indicates that the t groups R^(6B) replace t hydrogen atoms at any position on the phenyl ring.
 2. The compound or salt of claim 1, wherein m is 2, n is 2 or 4, R^(1L) is NH, R^(2L) is C, and R^(3L) is NH.
 3. The compound or salt of claim 1, wherein L¹ is a divalent linking group with a structure selected from an oligoamide which comprises a total of 1 to 5, more preferably a total of 1 to 3, and most preferably a total of 1 or 2 amide bonds within its backbone, and an oligo(ester-amide) which comprises a total of 2 to 5, more preferably a total of 2 to 3, and most preferably a total of 2 amide and ester bonds within its backbone, which linking group may carry a group EDS.
 4. The compound or salt of claim 2, wherein the moiety —X²-L¹-X¹— in formula (I) has a structure selected from: *—C(O)—NH—R⁷—NH—C(O)—R⁸—C(O)—NH—  (L-1), *—C(O)—NH—R^(9A)—NH—C(O)—R^(10A)—C(O)—NH—R^(11A)—NH—C(O)—  (L-2A), and *—C(O)—NH—R^(9B)—C(O)—NH—R^(10B)—C(O)—NH—R^(11B)—NH—C(O)—  (L-2B); wherein the amide bond marked with * is attached to the carbon atom carrying R² in formula (I), and wherein R⁷, R⁸, R^(9A), R^(9B), R^(11A) and R^(11B) are independently selected from optionally substituted C2 to C10 alkanediyl, which alkanediyl groups may each be substituted by one or more substituents independently selected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, —NHC(NH)NH₂, and a group EDS, and R^(10A) and R^(10B) are selected from optionally substituted C2 to C10 alkanediyl, and optionally substituted C6 to C10 arenediyl, which alkanediyl and arenediyl group may each be substituted by one or more substituents independently selected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, —NHC(NH)NH₂, and a group EDS.
 5. The compound or salt of claim 4, wherein the moiety —X²-L¹-X¹— has a structure selected from: *—C(O)—NH—CH(COOH)—R¹²—NH—C(O)—R¹³—C(O)—NH—  (L-3), *—C(O)—NH—CH(COOH)—R¹⁴—NH—C(O)—R¹⁵—C(O)—NH—R¹⁶—CH(COOH)—NH—C(O)—   (L-4), and *—C(O)—NH—CH(COOH)—R¹⁷—C(O)—NH—R¹⁸—C(O)—NH—R¹⁹—CH(COOH)—NH—C(O)—   (L-5); wherein the bond marked with * is attached to the carbon atom carrying R² in formula (I), R¹² and R¹⁴ are independently selected from linear C2 to C6 alkanediyl, R¹³ is a linear C2 to C10 alkanediyl, R¹⁵ and R¹⁶ are independently selected from linear C2 to C6 alkanediyl, and wherein each of R¹³ and R¹⁵ may carry one group EDS as a substituent, R¹⁷ is a linear C2 to C6 alkanediyl, R¹⁸ is a phenylene group, and R¹⁹ is a linear C2 to C6 alkanediyl.
 6. The compound or salt of claim 1, wherein R² is an optionally substituted aralkyl group selected from optionally substituted —CH₂-phenyl and optionally substituted —CH₂-naphthyl, wherein the phenyl and the naphthyl group are optionally substituted with a substituent selected from halogen, preferably I, and —OH.
 7. The compound or salt of claim 1, wherein R³ is an optionally substituted aralkyl group selected from optionally substituted —CH₂-phenyl and optionally substituted —CH₂-naphthyl, wherein the phenyl and the naphthyl group are optionally substituted with a substituent selected from halogen and —OH.
 8. The compound or salt of claim 1, wherein r is 1, and wherein R⁴ is selected from optionally substituted phenyl, optionally substituted naphthyl, and a group EDS, which phenyl group and naphthyl group are optionally substituted with a substituent selected from halogen, —OH and —NH₂.
 9. The compound or salt of claim 1, wherein X³ is an amide bond or a group of the formula

wherein the marked bond at the carbonyl group attaches X³ to R^(M) and the other marked bond attaches X³ to the remainder of the molecule.
 10. The compound or salt of claim 1, wherein R^(M) is a chelating group optionally containing a chelated non-radioactive or radioactive cation.
 11. The compound or salt of claim 1, wherein the compound of formula (I) either contains one group EDS which is carried by the linking group L¹, or contains two groups EDS, one being represented by R⁴ and one being carried by L¹.
 12. The compound or salt of claim 1, which contains a group EDS which has the formula (E-2A):

wherein

marks the bond which attaches the group EDS to the remainder of the compound of formula (I); and t is 1 or 2, and R^(6A) is selected from —NO₂ and —COOH.
 13. (canceled)
 14. A pharmaceutical or diagnostic composition comprising or consisting of one or more compounds or salts in accordance with claim
 1. 15. (canceled)
 16. A method for imaging or treating prostate-specific membrane antigen (PSMA)-associated cancer in a subject, comprising administering to the subject an effective amount of the composition of claim
 14. 17. The method of claim 16, wherein the chelated cation is ¹⁷⁷Lu.
 18. (canceled)
 19. (canceled)
 20. The method of claim 16, wherein the PSMA-associated cancer is glioma, lung cancer, or prostate cancer.
 21. (canceled)
 22. A method for treating prostate cancer in a subject, comprising administering to the subject an effective amount of a composition of claim
 14. 